Uses of conjugates of GM-CSF and IL-7 to treat viral infections

ABSTRACT

In certain embodiments, this disclosure relates to conjugates comprising GM-CSF and IL-7 and uses related thereto, e.g., enhancing the adaptive immune system. Typically the GM-CSF and IL-7 are connected by a polymer linker, e.g., polypeptide. In certain embodiments, the disclosure relates to nucleic acids encoding these polypeptide conjugates, vectors comprising nucleic acid encoding polypeptide conjugates, and protein expression systems comprising these vectors such as infectious viral particles and host cells comprising such a nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/355,673filed May 1, 2014 that granted as U.S. Pat. No. 9,375,465 on Jun. 28,2016, which is the National Stage of International Application No.PCT/US2012/064769 filed Nov. 13, 2012, which claims priority to U.S.Provisional Application No. 61/559,355 filed Nov. 14, 2011. The entiretyof each of these applications is hereby incorporated by reference forall purposes.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA THEOFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 11172USDIV-revised ST25.txt. The text file is 9KB, was created on Dec. 27, 2017, and is being submitted electronicallyvia EFS-web.

BACKGROUND

Although many viruses are cleared by the immune system, certainretroviruses, like HIV, evade the immune system, lie dormant, spread,and cause chronic infections. The World Health Organization estimatesthat AIDS has killed more than 25 million people since it was firstrecognized. In 2007, there were 2.7 million new HIV infections and 2million HIV-related deaths. Anti-retroviral drugs are medications forthe treatment of infection by retroviruses. When several antiviralagents are taken in combination with a retroviral drug, the approach isknown as highly active antiretroviral therapy (HAART). Although HAARTmay improve symptoms associated with infection, there is currently nocure for HIV. HAART can also have serious side-effects. Regimens can becomplicated, requiring patients to take several pills at various timesduring the day. If patients miss a dose, drug resistance can develop.Therefore, there remains a need for improved antiviral therapies. Inparticular, there remains a need for antiviral therapies with reducedtoxicity and improved efficacy over existing treatments.

Analogous to virus, cancer is thought to occur as a result of an immunesystem that is not properly removing uncontrolled proliferating cancercells. Stimulating the immune system to recognize and eliminatecancerous cells has become a promising strategy for therapeutictreatments. For example, Provenge™ is a FDA-approved autologous cellularimmunotherapy treatment. Peripheral blood leukocytes of a subject areharvested via leukapheresis. These enriched monocytes are incubatedprostatic acid phosphatase (PAP) conjugated to cytokine granulocytemacrophage colony stimulating factor (PAP-GM-CSF). GM-CSF is thought todirect the target antigen to receptors on DC precursors, which thenpresent PAP on their cell surface in a context sufficient to activate Tcells for the cells that express PAP. Activated, PAP presenting DCs areadministered to the subject to elicit an immune response retardingcancer growth. This strategy requires isolation and expansion of cellsof the subject, and typically treatment does not entirely clear thesubject of cancer or tumors. Thus, there is a need to identify improvedmethods.

Li et al., Clinical Immunology (2007) 123, 155-165, reported thatrecombinant IL-7 enhances the potency of GM-CSF-secreting tumor cellimmunotherapy. Pellegrini et al., Nature Medicine, 2009, 15, 528-536,reported that when combined with a GM-CSF-secreting tumor cellimmunotherapy, IL-7 prolonged the survival of tumor-bearing mice.Schroten-Loef et al., Cancer Immunol Immunother, (2009), 58:373-381,disclose a prostate cancer vaccine comprising whole cells secretingIL-7.

Williams and Park, Cancer, 1991, 67(10 Suppl):2705-7, disclose a fusionprotein of the granulocyte-macrophage colony-stimulating factor (GM-CSF)and interleukin-3 (IL-3). See also WO 2005/0053579, WO 2005/026820, WO2008/0014612, and U.S. Pat. Nos. 7,323,549 and 7,217,421.

SUMMARY

In certain embodiments, this disclosure relates to conjugates comprisinga polypeptide of GM-CSF and a polypeptide IL-7. Typically the GM-CSF andIL-7 are connected by a polymer linker, e.g., polypeptide. In certainembodiments, the disclosure relates to nucleic acids encoding thesepolypeptide conjugates, vectors comprising nucleic acid encodingpolypeptide conjugates, and protein expression systems comprising thesevectors such as infectious viral particles and host cells comprisingsuch a nucleic acids.

In certain embodiments, the disclosure relates to pharmaceuticalcompositions comprising conjugates and vectors disclosed herein and apharmaceutically acceptable excipient. In certain embodiments, thedisclosure relates to vaccines comprising conjugates and vectorsdisclosed herein and an antigen and optionally an adjuvant. Typically,the antigen is live attenuated virus, killed virus, a virus-likeparticle, virosome, cancerous cell, lipid bilayer structure with asurface antigen, viral protein or glycoprotein, bacteria, or bacterialantigen, or tumor associated antigen. In certain embodiments, theantigen is conjugated to a dendritic cell marker.

In certain embodiments, the disclosure relates to methods of treating orpreventing a viral, bacterial, or parasitic infection comprisingadministering an effective amount of a pharmaceutical compositioncomprising a conjugate or vector disclosed herein optionally incombination with an antigen and optionally an adjuvant. In certainembodiments, the subject is at risk or, exhibiting symptoms of, ordiagnosed with a viral infection, such as a chronic viral infection.

In certain embodiments, the disclosure relates to methods of treating orpreventing a viral infection comprising administering an effectiveamount of a vaccine comprising a conjugate disclosed herein to a subjectin need thereof.

In certain embodiments, the subject is diagnosed with influenza A virusincluding subtype H1N1, influenza B virus, influenza C virus, rotavirusA, rotavirus B, rotavirus C, rotavirus D, rotavirus E, SARS coronavirus,human adenovirus types (HAdV-1 to 55), human papillomavirus (HPV) Types16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, parvovirus B19,molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cellpolyomavirus, coxsackie A virus, norovirus, Rubella virus, lymphocyticchoriomeningitis virus (LCMV), yellow fever virus, measles virus, mumpsvirus, respiratory syncytial virus, rinderpest virus, Californiaencephalitis virus, hantavirus, rabies virus, ebola virus, marburgvirus, herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2),varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus(CMV), herpes lymphotropic virus, roseolovirus, or Kaposi'ssarcoma-associated herpesvirus, hepatitis A, hepatitis B, hepatitis C,hepatitis D, hepatitis E or human immunodeficiency virus (HIV).

In certain embodiments, the disclosure relates to administering aconjugate or vector disclosed herein in combination with anotherantiviral agent such as abacavir, acyclovir, acyclovir, adefovir,amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla,boceprevir, cidofovir, combivir, darunavir, delavirdine, didanosine,docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir,famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet,ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir,inosine, interferon type III, interferon type II, interferon type I,lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone,nelfinavir, nevirapine, nexavir, oseltamivir, peginterferon alfa-2a,penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir,ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, stavudine,tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir,tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc,vidarabine, viramidine zalcitabine, zanamivir, and/or zidovudine.

In certain embodiments, the disclosure relates to methods of treating orpreventing cancer comprising administering a pharmaceutical compositioncomprising a conjugate or vector disclosed herein to a subject in needthereof.

In certain embodiments, the disclosure relates to methods of treating orpreventing cancer comprising administering autologous blood cellsactivated with a cancer antigen conjugated to GM-CSF in combination witha conjugate disclosed herein to a subject in need thereof.

In certain embodiments, the disclosure relates to methods of activatingperipheral blood cells comprising mixing peripheral blood cells with aconjugate disclosed herein comprising a tumor associated antigen/cancermarker under conditions such that increase expression of CD54 occurs. Incertain embodiments, the disclosure relates to product produced bymixing peripheral blood cells and with a conjugate disclosed hereinunder conditions such that increase expression of CD54 occurs. Incertain embodiments, the disclosure relates to methods of treating orpreventing cancer comprising administering an effective amount of aproduct made by mixing peripheral blood cells with a conjugate disclosedherein to subject from whom the peripheral blood cells were obtained.

In some embodiments, the disclosure relates to a method of treating orpreventing cancer comprising by administering a pharmaceuticalcomposition comprising conjugates or vector disclosed herein to asubject diagnosed with, exhibiting symptoms of, or at risk of cancerwherein the cancer is a hematological malignancy such as a leukemia orlymphoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia(AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma(SLL), chronic myelogenous leukemia, acute monocytic leukemia (AMOL),Hodgkin's lymphomas, and non-Hodgkin's lymphomas such as Burkittlymphoma, B-cell lymphoma and multiple myeloma. Other contemplatedcancers include cervical, ovarian, colon, breast, gastric, lung, skin,ovarian, pancreatic, prostate, head, neck, and renal cancer.

Within any of the cancer management methods disclosed herein, theconjugate or vector may be administered in combination with ananti-cancer agent such as gefitinib, erlotinib, docetaxel, cis-platin,5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate,cytosine arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin,daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin andmithramycin, vincristine, vinblastine, vindesine, vinorelbine taxol,taxotere, etoposide, teniposide, amsacrine, topotecan, camptothecin,bortezomib, anagrelide, tamoxifen, toremifene, raloxifene, droloxifene,iodoxyfene, fulvestrant, bicalutamide, flutamide, nilutamide,cyproterone, goserelin, leuprorelin, buserelin, megestrol, anastrozole,letrozole, vorazole, exemestane, finasteride, marimastat, trastuzumab,cetuximab, dasatinib, imatinib, bevacizumab, combretastatin,thalidomide, and/or lenalidomide or combinations thereof.

In certain embodiments, the disclosure relates to gene therapiescomprising administering vectors comprising nucleic acid encodingconjugates disclosed herein to a subject in need thereof.

In certain embodiments, the disclosure contemplates incorporatingconjugates disclosed herein into the surfaces of particles, e.g., cells,liposomes, micelles, vesicles, bilayer structures, virosomes, andvirus-like particles. The conjugates may be linked to lipophilicmoieties, e.g., fatty acids and GPI. In one example, the disclosurecontemplates a GPI anchored conjugate comprising GPI, GM-CSF, IL-7, andoptionally an antigen, adjuvant, or other polypeptide. It iscontemplated that these particles may contain other surfacepolypeptides, antigens and co-stimulatory molecules such as B7-1, B7-2,ICAM-1, and/or IL-2. It is contemplated that these particles may be usedin all the applications conjugates disclosed herein are mentioned.

Within certain embodiments, any of the conjugates disclosed herein maybe further conjugated to an adjuvant, cytokine, co-stimulatory molecule,antigen, protein, or glycoprotein. In certain embodiments, the antigenis a viral protein or a cancer marker.

In certain embodiments, the cancer marker is selected from PAP(prostatic acid phosphatase), prostate-specific antigen (PSA), (PSMA)prostate-specific membrane antigen, early prostate cancer antigen-2(EPCA-2), AKAP-4 (A kinase [PRKA] anchor protein 4), NGEP (new geneexpressed in prostate), PSCA (prostate stem cell antigen), STEAP(six-transmembrane epithelial antigen of the prostate), MUC 1 (mucin 1),HER-2, BCL-2, MAGE antigens such as CT7, MAGE-A3 and MAGE-A4, ERK5,G-protein coupled estrogen receptor 1, CA15-3, CA19-9, CA 72-4, CA-125,carcinoembryonic antigen, CD20, CD31, CD34, PTPRC (CD45), CD99, CD117,melanoma-associated antigen (TA-90), peripheral myelin protein 22(PMP22), epithelial membrane proteins (EMP-1, -2, and -3), HMB-45antigen, MART-1 (Melan-A), S100A1, and S100B or fragments or mutatedforms thereof.

In certain embodiments, the viral antigen is selected from an influenzavirus hemagglutinin and neuraminidase; cytomegalovirus glycoprotein gB,p28, p38, p50, p52, p65, and p150; Borrelia p41; HIV nef, integrase,gag, protease, tat, env, p31, p17, p24, p31, p55, p66, gp32, gp36, gp39,gp41, gp120, and gp160; SIV p55; HBV core, surface antigen, andaustralian antigen; HCV core nucleocapsid, NS3, NS4, and NS5; Dengue envand NS1; EBV early antigen, p18, p23, gp125, nuclear antigen (EBNA)-1,EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP), latentmembrane proteins (LMP)-1, LMP-2A and LMP-2B; and herpes simplex virusgD and gG or fragments or mutated forms thereof.

In certain embodiments, the adjuvant or cytokine is selected from IL-2,IL-12, IL-15, IL-18, IL-21, IL-27, IL-31, IFN-alpha, flagellin,unmethylated, CpG oligonucleotide, lipopolysaccharides, lipid A, andheat stable antigen (HSA).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the construction of a GM-CSF and IL-7 conjugate referredto as GIFT7-linker italicized. Amino acid sequence of mGIFT7 (SEQ IDNO:2) and hGIFT7 (SEQ ID NO:1). Schematic representation of the fusionGIFT7 showing GM-CSF at the N-terminus linked to C-terminal domain IL-7by peptide linker.

FIG. 1B shows denatured immunoblotting using supernatant derived fromGIFT7 or mock-transfected 293 cells. rGM-CSF or rIL-7 were used ascontrols. The blot was probed with polyclonal goat anti-IL-7 andanti-GM-CSF antibody to detect fusion protein secretion.

FIG. 2A shows GIFT7-mediated biochemical and cellular effects onlymphoid cells. GIFT7-mediated STATS phosphorylation of primary T cellsand RAW247.6 cells upon stimulation.

FIG. 2B shows data where 1.25×10⁵ per well of CD3/CD28 pre-activated Tcells in 96 well plates were stimulated with GIFT7, GM-CSF, IL-7,GM-CSF+IL-7 or media alone for 6 days. Cell viability and proliferationwere assessed by trypa blue exclusion and CFSE dilution, respectively.*P<0.05

FIG. 2C shows data where percent apoptotic cells were assessed byPI/Annexin V co-staining.

FIG. 3A shows data on GIFT7-mediated conversion of CD8 T cells.Immunophenotype of GIFT7-treated pre-activated T cells. Ex vivomanipulated T cells were stained with the indicated antibodies andsurface expression was analyzed by FCAS.

FIG. 3B shows data on GIFT7-mediated conversion of CD8 T cells.

FIG. 3C shows data on IFNγ production from responding GIFT7OT1-CD8 Tcells, reactivated in vitro by OVA-pulsed macrophages in the presence orabsence of TGF-β. Results represent the mean value of three independentexperiments+/−SD; *P→0.05

FIG. 3D shows data where 5×10⁶ GIFT7OT1-CD8 T cells were administeredinto WT naïve pep-boy mice, which were subsequently injected withrecombinant OVA after 21 days. 7 days later, splenic content wasanalyzed for the frequency of exogenous OT1 cells.

FIG. 3E shows data where two doses of 10×10⁶ GIFT7 OT1-CD8 T cells weregiven in vivo to mice with palpable EG7.

FIG. 4A shows the effect of GIFT7 on thymic output. Thymoctes weretreated with GIFT7 or cytokine control in vitro for 5 days. Ex vivomanipulated thymocytes were stained with the indicated antibodies andsurface expression was analyzed by FCAS.

FIG. 4B shows the effect of GIFT7 on thymic output.

FIG. 4C shows data for CFSE-labeled CD4/CD8 depeleted thymocytes wereassessed by FCAS with co-staining of γδTCR after 5 days of in vitroGIFT7 treatment.

FIG. 4D shows data for whole thymic output of naïve mice administeredwith GIFT7 at the indicated doses was analyzed at day 7.

FIG. 5A shows the effect of GIFT7 on NHP and human PBMC. PBMC wasobtained from healthy primates and treated with GIFT7 after 48 hours ofin vitro activation with conA.

FIG. 5B shows data when PBMC was obtained from healthy volunteers andsubsequently treated with GIFT7 after 48 hours of in vitro activationwith CD3/CD28. Ki67 and PD1 expression was assessed by FACS.

FIG. 6 shows data indicating GIFT7 leads to enhanced T cellproliferation.

FIG. 7A illustrates a method.

FIG. 7B shows data on total cells.

FIG. 7C shows data at days 3 and 7.

FIG. 7D shows data indicating Simian PBMC (sPBMC) treated with hGIFT7are Ki67^(hi).

FIG. 7E shows data indicating Simian PBMC (sPBMC) treated with hGIFT7are PD1^(low).

FIG. 8A shows data indicating GIFT7 leads to the generation of centralmemory-like T cells post-activation.

FIG. 8B shows data.

FIG. 8C shows a method and data.

FIG. 9A shows data indicating a GIFT7-mediated anti-tumor effect invivo.

FIG. 9B shows fraction survival v. days pots-AML challenge.

FIG. 10A shows data indicating GIFT7 leads to transient thymichyperplasia in young mice where 2-month-old C57Bl/6 were injected withthree doses of GIFT7 or IL7 i.v. (5 ug/Kg) at 1-day interval. Thymicollected on day 7, 14, or 35 were analyzed for total and subsetcellularity.

FIG. 10B shows total thymic cellularity in GIFT7 or IL7-treated groupsat each time point. Results represent the mean cell number+/−SD (n=3-5);*P<0.05 (

FIG. 10C shows data when dissociated thymi were analyzed for CD3, CD4,CD8, CD25, and CD44 expression. Dot plots from one representative animalindicate the frequency of each thymic subset on day 7.

FIG. 10D shows data for day 14. The histograms represent the number ofcells associated with each phenotype. Data represent mean+/−SD (n=5)*p<0.05.

FIG. 11A shows data indicates that GIFT7 corrects age-related thymicatrophy. Hematoxylin- and eosin-stained paraffin sections of thymus fromone representative GIFT7- or IL7-treated aged mice. Higher magnificationshow cortical hyperplasia infiltrating the lining of cortical medullaryjunction (solid arrow) compared to the smooth lining in IL7-treatedgroup (interrupted arrow). Asterisk, adipose tissue deposit; C, cortex;M, medulla. The histogram indicates the ratio of cortical/medullarythickness. Data represent the mean value+/−SD (n=6); *p<0.05

FIG. 11B shows data indicating an increase in the number of total, DN,DP, SPCD4, and SPCD8 thymocytes of the GIFT7-treated aged mice. Thymiwere dissociated and analyzed by flow cytometry. Histogram representmean number of cells+/−SD (n=6).

FIG. 11C shows data indicating GIFT7 administration leads to increasedthymic output. mRNA expression of single-joint (sj) TREC to TCRα ratioof splenocytes from aged mice were measured by RT-PCR. Histogramrepresents fold difference of the relative mRNA expression (2-ΔCT ofsjTREC to TCRα) from each treated mice normalized to the mean relativemRNA expression from the untreated group. RT-PCR was performed intriplicates.

FIG. 12A shows data indicating CD44^(int) DN resident thymocytes drivesGIFT7-mediated reconstitution. Representative flow cytometry plotsindicate GIFT7-mediated expansion in the frequency of CD44^(int)CD25⁻ DNthymocytes. Number represents the percentage in each gated region.

FIG. 12B shows data indicating an increase in the number of total, DN4and both CD44^(int) and CD44^(hi) DN1 in GIFT7-treated aged mice. Thymiwere dissociated and analyzed by flow cytometry. Histogram representsmean number of cells+/−SD (n=6).

FIG. 12C shows data indicating IL7Rα expression is primarily located inthe DN1 CD44^(hi) subset. Thymi derived from GIFT7-, IL7-, or untreatedaged mice were dissociated and analyzed by surface expression of CD4,CD8, CD25, CD44, and CD127 (IL7Rα) by flow cytometry. Histogramrepresents IL7Rα expression in different subsets of DN thymocytes.

FIG. 13A shows a schematic representation indicates 7 i.p. injections ofGIFT7 or cytokine controls at 5 ug/kg (arrowhead) in aged mice. 2.5×10⁴PFU of MCMV were injected 6 days after the treatment. Spleens wereanalyzed for total or viral-specific cellularity 10 days post-infection.Data indicates GIFT7 treatment enhances anti-CMV CD8 response in agedmice.

FIG. 13B shows a representative flow dot plots show the CMV-specific CTLresponse in young or aged mice treated with different treatment. Numbersindicate the percentage of CMV+CTL.

FIG. 13C shows a histogram represents the mean percentage.

FIG. 13D shows number of cells in each group+/−SD (n=4).

DETAILED DESCRIPTION

GM-CSF-IL-7 Fusokine (GIFT7)

Interleukin-7 (IL-7) is a γ-chain cytokine that plays a role in T celldevelopment and homeostasis by signaling through its cognate receptor,IL-7R or CD127, and inducing T cell survival and/or proliferation.Disclosed herein is a conjugate protein comprising GM-CSF linked to IL-7by a peptide linker, and this fusion cytokine (fusokine) transgene canbe expressed and secreted by mammalian cell lines in a manner that isrecognized by both anti-GM-CSF and anti-IL-7 antisera (FIG. 1). Thisfusokine is very potent in mediating rejection of cancer by immunecompetent recipients. GIFT7 has effects on antigen experienced CD8T-cells, converting them into Central Memory Phenotype, GIFT7 CD8 (FIG.3). It is important to note that a GM-CSF/IL3 fusion does not displaygain of function, i.e., a GM-CSF/interleukin fusion will not necessarilyacquire this function. Also, the gain of function seen with GM-CSF/IL-2fusion affected NK cells solely whilst GIFT7 specifically enhances thebiology of memory T-cells, double negative thymocytes and expands CD4t-cells as well.

GIFT7 has novel pharmacological properties distinct from IL-7 alone.Specifically, (1) GIFT7 leads to a hyperagonistic response manifest byincrease pSTAT5 in murine CD127⁺ lymphoid cells leading to a massiveproliferative and pro-survival effect (FIG. 2); (2) The cell biologicalconsequence on murine splenic T-cells is to selectively expand anantigen-experienced CD8⁺ subset with a Central Memory (CM) phenotype(FIG. 3A,B); (3) GIFT7 CD8 also demonstrate significant Th1-IFNγ-drivenanti-tumor response in vitro and in vivo, and prolonged in vivopersistence following adoptive transfer in normal or tumor-bearing mice(FIG. 3C,D,E); (4) GIFT7 can expand CD4⁻CD8⁻CD44⁺CD25⁻ double negative 1(DN1) T cell precursors in both αβ and γδ T cell lineage (FIG. 4A,B,C);The direct administration of GIFT7 protein to normal mice leads to theexpansion of double negative (DN) and CD8⁺ thymic subsets (FIG. 4D); and(5) the unique expansive effect of GIFT7 was also demonstrate onperipheral blood mononuclear cells (PBMC) derived from non-humanprimates (NHP) and human, in that GFIT7 stimulation of pre-activatedPBMC typically leads to T cells proliferation without exhaustion(Ki67^(hi) and PD1^(low)) compared to monomeric cytokine IL-7 (FIG. 5A,B).

Data collected on GIFT7 and GIFT7-CD8 suggests use in the treatment ofhuman disorders where an enhanced of immune response is desirable, inparticular chronic infectious ailments and cancer. Moreover, based onobservations of GIFT7-mediated enhancement on thymic output (inparticular in the DN1, γδ T cell precursor and SPCD8), GIFT7 orGIFT7-enhanced T cell precursors may be use of for the treatment of anon-exclusive listing of human immune deficient ailments such ascongenital or HIV-mediated acquired immunodeficiency, after chemotherapyor after-hematopoietic stem cell transplant (HSCT) and for the ageing.

The present disclosure encompasses fusion proteins involving full-lengthpre-processed forms, as well as mature processed forms, fragmentsthereof and variants of each or both of the GM-CSF and IL-7 entities,including allelic as well as non-naturally occurring variants. Inaddition to naturally-occurring allelic variants of the GM-CSF and IL-7entities that may exist in the population, the skilled artisan willfurther appreciate that changes (i.e. one or more deletions, additionsand/or substitutions of one or more amino acid) can be introduced bymutation using classic or recombinant techniques to effect random ortargeted mutagenesis. A suitable variant in use in the presentdisclosure typically has an amino acid sequence having a high degree ofhomology with the amino acid sequence of the corresponding nativecytokine. In one embodiment, the amino acid sequence of the variantcytokine in use in the fusion protein of the disclosure is at least 70%,at least about 75%, at least about 80%, at least about 90%, typically atleast about 95%, more typically at least about 97% and even moretypically at least about 99% identical to the corresponding nativesequence. In certain embodiments such native sequence is of human GM-CSFand/or human IL-7.

Percent identities between amino acid or nucleic acid sequences can bedetermined using standard methods known to those of skill in the art.For instance for determining the percentage of homology between twoamino acid sequences, the sequences are aligned for optimal comparisonpurposes. The amino acid residues at corresponding amino acid positionsare then compared. Gaps can be introduced in one or both amino acidsequence(s) for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes. When a position in the firstsequence is occupied by the same amino acid residue as the correspondingposition in the second sequence, then the sequences are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps which need to be introduced foroptimal alignment and the length of each gap. The comparison ofsequences and determination of percent identity and similarity betweentwo sequences can be accomplished using a mathematical algorithm (e.g.Computational Molecular Biology, 1988, Ed Lesk A M, Oxford UniversityPress, New York; Biocomputing: Informatics and Genome Projects, 1993, EdSmith D. W., Academic Press, New York; Computer Analysis of SequenceData, 1994, Eds Griffin A. M. and Griffin H. G., Human Press, NewJersey; Sequence Analysis Primer, 1991, Eds Griskov M. and Devereux J.,Stockton Press, New York). Moreover, various computer programs areavailable to determine percentage identities between amino acidsequences and between nucleic acid sequences, such as GCG™ program(available from Genetics Computer Group, Madison, Wis.), DNAsis™ program(available from Hitachi Software, San Bruno, Calif.) or the MacVector™program (available from the Eastman Kodak Company, New Haven, Conn.).

Suitable variants of GM-CSF and IL-7 entities for use in the presentdisclosure are biologically active and retain at least one of theactivities described herein in connection with the correspondingpolypeptide. Typically, the therapeutic effect (e.g. anti-tumoractivity, by-pass of tumor-induced immune energy) is preserved, althougha given function of the polypeptide(s) may be positively or negativelyaffected to some degree, e.g. with variants exhibiting reducedcytotoxicity or enhanced biological activity. Amino acids that areessential for a given function can be identified by methods known in theart, such as by site-directed mutagenesis. Amino acids that are criticalfor binding a partner/substrate (e.g. a receptor) can also be determinedby structural analysis such as crystallization, nuclear magneticresonance and/or photoaffinity labeling. The resulting variant can betested for biological activity in assays such as those described above.

For example, in one class of functional variants, one or more amino acidresidues are conservatively substituted. A “conservative amino acidsubstitution” is one in which the amino acid residue in the nativepolypeptide is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. Typically, substitutions are regarded asconservative when the replacement, one for another, is among thealiphatic amino acids Ala, Val, Leu, and Ile; the hydroxyl residues Serand Thr; the acidic residues Asp and Glu; the amide residues Asn andGln; the basic residues Lys and Arg; or the aromatic residues Phe andTyr. Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a cytokine coding sequence, such as bysaturation mutagenesis, and the resultant mutant can be screened for itsbiological activity as described herein to identify mutants that retainat least therapeutic activity.

Although the GM-CSF and IL-7 entities can be directly fused in thefusion protein of the disclosure, it is however typical to use a linkerpeptide for joining GM-CSF and IL-7. The purpose of the linker is toallow the correct formation, folding and/or functioning of each of theGM-CSF and IL-7 entities. It should be sufficiently flexible andsufficiently long to achieve that purpose. Typically, the codingsequence of the linker may be chosen such that it encouragestranslational pausing and therefore independent folding of the GM-CSFand IL-7 entities. A person skilled in the art will be able to designsuitable linkers in accordance with the disclosure. The presentdisclosure is, however, not limited by the form, size or number oflinker sequences employed. Multiple copies of the linker sequence ofchoice may be inserted between GM-CSF and IL-7. The only requirement forthe linker sequence is that it functionally does not adversely interferewith the folding and/or functioning of the individual entities of thefusion protein. For example, a suitable linker is 5 to 50 amino acidlong and may comprise amino acids such as glycine, serine, threonine,asparagine, alanine and proline (see for example Wiederrecht et al.,1988, Cell 54, 841; Dekker et al., 1993, Nature 362, 852; Sturm et al.,1988, Genes and Dev. 2, 1582; Aumailly et al., 1990 FEBS Lett. 262, 82).Repeats comprising serine and glycine residues are typical in thecontext of the disclosure. Specific examples of suitable linkersconsists of two or three or more (e.g. up to eight) copies of thesequence Gly-Gly-Gly-Gly-Ser (GGGGS). It will be evident that thedisclosure is not limited to the use of these particular linkers.

The disclosure further includes fusion proteins which comprise, oralternatively consist essentially of, or alternatively consist of anamino acid sequence which is at least 70%, 75%, 80%, 90%, 95%, 97%, 99%homologous or even better 100% homologous (identical) to all or part ofany of the amino acid sequences recited in SEQ ID NO: 1-2.

In the context of the present disclosure, a protein “consists of” anamino acid sequence when the protein does not contain any amino acidsbut the recited amino acid sequence. A protein “consists essentially of”an amino acid sequence when such an amino acid sequence is presenttogether with only a few additional amino acid residues, typically fromabout 1 to about 50 or so additional residues. A protein “comprises” anamino acid sequence when the amino acid sequence is at least part of thefinal (i.e. mature) amino acid sequence of the protein. Such a proteincan have a few up to several hundred additional amino acids residues.Such additional amino acid residues can be naturally associated witheach or both entities contained in the fusion or heterologous aminoacid/peptide sequences (heterologous with respect to the respectiveentities). Such additional amino acid residues may play a role inprocessing of the fusion protein from a precursor to a mature form, mayfacilitate protein trafficking, prolong or shorten protein half-life orfacilitate manipulation of the fusion protein for assay or production,among other things. Typically, the fusion proteins of the disclosurecomprise a signal peptide at the NH2-terminus in order to promotesecretion in the host cell or organism. For example, the endogenoussignal peptide (i.e. naturally present in the cytokine present at theNH2 terminus of said fusion) can be used or alternatively a suitableheterologous (with respect to the cytokine in question) signal peptidesequence can be added to the cytokine entity present at the NH2 terminusof the fusion or inserted in replacement of the endogenous one.

In the context of the disclosure, the fusion proteins of the disclosurecan comprise cytokine entities of any origin, i.e. any human or animalsource (including canine, avian, bovine, murine, ovine, feline, porcine,etc). Although “chimeric” fusion proteins are also encompassed by thedisclosure (e.g. one cytokine entity of human origin and the other of ananimal source), it is typical that each entity be of the same origin(e.g. both from humans).

The fusion proteins of the present disclosure can be produced bystandard techniques. Polypeptide and DNA sequences for each of thecytokines involved in the fusion protein of the present disclosure arepublished in the art, as are methods for obtaining expression thereofthrough recombinant or chemical synthetic techniques. In anotherembodiment, a fusion-encoding DNA sequence can be synthesized byconventional techniques including automated DNA synthesizers. Then, theDNA sequence encoding the fusion protein may be constructed in a vectorand operably linked to a regulatory region capable of controllingexpression of the fusion protein in a host cell or organism. Techniquesfor cloning DNA sequences for instance in viral vectors or plasmids areknown to those of skill in the art (Sambrook et al, 2001, “MolecularCloning. A Laboratory Manual”, Laboratory Press, Cold Spring HarborN.Y.). The fusion protein of the disclosure can be purified from cellsthat have been transformed to express it.

The present disclosure also provides a nucleic acid molecule encodingthe fusion protein of the disclosure. Within the context of the presentdisclosure, the term “nucleic acid” and “polynucleotide” are usedinterchangeably and define a polymer of nucleotides of any length,either deoxyribonucleotide (DNA) molecules (e.g., cDNA or genomic DNA)and ribonucleotide (RNA) molecules (e.g., mRNA) and analogs of the DNAor RNA generated using nucleotide analogs (see U.S. Pat. No. 5,525,711and U.S. Pat. No. 4,711,955 as examples of nucleotide analogs). Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. The sequence of nucleotides mayalso be interrupted by non-nucleotide elements. The nucleic acidmolecule may be further modified after polymerization, such as byconjugation with a labeling component. The nucleic acid, especially DNA,can be double-stranded or single-stranded, but typically isdouble-stranded DNA. Single-stranded nucleic acids can be the codingstrand (sense strand) or the non-coding strand (anti-sense strand).

The nucleic acid molecules of the disclosure include, but are notlimited to, the sequence encoding the fusion protein alone, but maycomprise additional non-coding sequences, for example introns andnon-coding 5′ and 3′ sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and mRNA stability. For example, the nucleic acid molecule ofthe disclosure can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb or 0.1 kb of nucleotide sequences which naturally flank (i.e.sequences located at the 5′ and 3′ ends) or are present within thegenomic DNA encoding GM-CSF and IL-7 entities.

According to a typical embodiment, the present disclosure providesnucleic acid molecules which comprise, or alternatively consistessentially of, or alternatively consist of a nucleotide sequenceencoding all or part of an amino acid sequence encoding a fusion proteinwhich is at least about 70%, at least about 75%, at least about 80%, atleast about 90%, at least about 95%, typically at least about 97%, moretypically at least about 99% homologous or even more typically 100%homologous to any of the amino acid sequences shown in SEQ ID NO: 1-2.

In another embodiment, a nucleic acid molecule of the disclosurecomprises a nucleic acid molecule which is a complement of all or partof a nucleotide sequence encoding the fusion protein shown in any of SEQID NO: 1-2. A nucleic acid molecule which is complementary to thenucleotide sequence of the present disclosure is one which issufficiently complementary such that it can hybridize to thefusion-encoding nucleotide sequence under stringent conditions, therebyforming a stable duplex. Such stringent conditions are known to thoseskilled in the art. A typical, non-limiting example of stringenthybridization conditions are hybridization in 6 times sodiumchloride/sodium citrate (SSC) at about 45 C, followed by one or morewashes in 0.2 times SSC, 0.1% SDS at 50-65 C. In one embodiment, thedisclosure pertains to antisense nucleic acid to the nucleic acidmolecules of the disclosure. The antisense nucleic acid can becomplementary to an entire coding strand, or to only a portion thereof.

In still another embodiment, the disclosure encompasses variants of theabove-described nucleic acid molecules of the disclosure e.g., thatencode variants of the fusion proteins that are described above. Thevariation(s) encompassed by the present disclosure can be created byintroducing one or more nucleotide substitutions, additions and/ordeletions into the nucleotide sequence by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Followingmutagenesis, the variant nucleic acid molecule can be expressedrecombinantly as described herein and the activity of the resultingprotein can be determined using, for example, assays described herein.Alternatively, the nucleic acid molecule of the disclosure can bealtered to provide preferential codon usage for a specific host cell(for example E. coli; Wada et al., 1992, Nucleic Acids Res. 20,2111-2118). The disclosure further encompasses nucleic acid moleculesthat differ due to the degeneracy of the genetic code and thus encodefor example the same fusion protein as any of those shown in SEQ ID NO:1-2.

Another embodiment of the disclosure pertains to fragments of thenucleic acid molecule of the disclosure, e.g. restriction endonucleaseand PCR-generated fragments. Such fragments can be used as probes,primers or fragments encoding an immunogenic portion of the fusionprotein.

The nucleic acid molecules of the present disclosure can be generatedusing the sequence information provided herein. The nucleic acidencoding each of the GM-CSF and IL-7 entities can be cloned or amplifiedusing cDNA or, alternatively, genomic DNA, as a template and appropriateprobes or oligonucleotide primers according to standard molecularbiology techniques (e.g., as described in Sambrook, et al. “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001) or standard PCR amplification techniquesbased on sequence data accessible in the art (such as those providedabove in connection with the fusion proteins of the disclosure or thoseprovided in the Examples part). Fusing of the GM-CSF sequence to theIL-7 sequence may be accomplished as described in the Experimental belowor by conventional techniques. For example, the GM-CSF and IL-7 encodingsequences can be ligated together in-frame either directly or through asequence encoding a peptide linker. The GM-CSF-encoding sequence canalso be inserted directly into a vector which contains the IL-7-encodingsequence, or vice versa. Alternatively, PCR amplification of the GM-CSFand IL-7-encoding sequences can be carried out using primers which giverise to complementary overhangs which can subsequently be annealed andre-amplified to generate a fusion gene sequence.

GIFT7 Overcomes T Cell Exhaustion Leading to the Expansion of PeripheralT Cell in a PD1^(low) State

T-cell based immunotherapy represents a promising strategy for chronicviral infections or malignancies. However, its efficacy depends on theinfused T cell fitness in vivo. Indeed, T cell exhaustion via theupregulation of PD-1 reflects the maladaptive immune response as a causeof effector cell failure. To address this issue, data herein indicatesthat GIFT7 leads to a hyperagonistic response manifest by increasedpSTAT5 and proliferation in CD3⁺/CD127⁺ cells. GIFT7 also selectivelyexpand a CD8⁺ subset from pre-activated T cells with a Central Memoryphenotype defined as CD8⁺CD44⁺CD62L⁺CCR7⁺KLRG⁻CD27⁺PD-1⁻ hereafterT_(GIFT7). Adoptive transfer of OT1-derived CD8 T_(GIFT7) intoOVA-EG7-bearing mice leads to a significant anti-tumor effect.Furthermore, T_(GIFT7) persist long-term whilst IL2-activated CD8T-cells are undetected. The unique expansive effect of the humanortholog of GIFT7 on peripheral blood mononuclear cells (PBMC) derivedfrom non-human primates (NHP) and humans, in that GIFT7 stimulation ofpre-activated PBMC leads to T cell prolifetaion without exhaustion(KI67^(hi) and PD-1^(low)) in CD4⁺, CD8⁺, and CD4⁻CD8⁻ compartments.This observation supports the use of GIFT7 primed T cells for chronicviral infections or malignancies where an inadequate T cell response isa common feature.

GIFT7 Reverses Age-Related Thymic Atrophy by Expanding CD44^(int)Double-Negative Resident Thymic Progenitors Via IL7Rα/γ_(c)Hypersignalling

Immune insufficiency secondary to ageing predisposes the host todetrimental infections. Immune senescence manifested by thymicinvolution correlates to the progressive decline in T cell receptor(TCR) repertoire and numerous defects in cellular immunity with severeclinical complications such as chronic inflammation, autoimmunediseases, and low vaccination efficacy. Normal thymopoiesis arises frommarrow-derived CD4⁻CD8⁻ double-negative T cell progenitors (DN).Thereafter, DN develop into mature single-positive (SP) CD4 or CD8 Tcells after expressing both CD4 and CD8 (double-positive-DP)transiently, leading to de novo T cell production. Age-relatedprogressive decline in thymic activity starts as early as the first yearof postnatal life and is due to a combination of reduced thymic stromaand intrathymic proliferation of lymphoid precursors.

Interleukin-7 (IL-7) plays a role in T cell development and homeostasisby signalling through its cognate receptor complex IL7Rα/γ_(c). Thecorrelation between age-related hypotonic thymic activity and IL7availability in the stromal niche is not clearly defined. IL7 isdetectable in aged thyme. The use of exogenous IL7 for therapeuticlymphogenesis has been attempted. However, peripheral T cellcompartmentalization—preferential expansion of naïve and central memoryCD4⁺ T cell—is a much more pronounced effect secondary to exogenous IL7administration; thymic function remains largely unaltered. See Chu etal., Blood, 2004, 104(4):1110-1119. The intrathymic implantation ofgenetically engineered IL7-producing stromal cells show no benefits inovercoming age-associated atrophy and the maintenance of peripheral Tcell pool despite a transient increase in intrathymic proliferation. SeePhillips et al., J Immunol, 2004, 173(8):4867-4874.

Part of the reason for its modest therapeutic effect is believed to berelated to the homeostatic nature of IL7/IL7Rα interaction. Theexpression of IL7Rα is tightly regulated: it is upregulated onhematolymphoid cells destined to persist; it is downregulated onquiescent or exhausted cells, and it is internalized upon ligandbinding. Thus, the in vivo use of IL7 for regenerative immunotherapy islimited by the requirement to achieve supraphysiological concentrationin order to overcome immune checkpoints to its activating effects.

Studies herein indicate that engineered fusion GIFT7 delivershyperagonistic signalling to IL7Rα/γ_(c), hyperphosphorylating signaltransducer and activator of transcription (STAT) 5. The biologicalconsequence of STATS hyperactivation is the induction of SPCD8 andCD44⁺CD25⁻ DN1 expansion in thymocyte culture. Systemic administrationof GIFT7 in young immune competent mice leads to an immediate increasein the number of DN1, which subsequently progress to an increase in thetotal number of thymocytes and DP. GIFT7-mediated hypertrophic effect issignificantly more pronounced in aged thymi; it precipitates theexpansion of a subset of early thymic precursors CD44int DN1, which iscapable of participating in normal T cell development, leading to asignificant enhancement of thymic cellularity, cortical hyperplasia, andperipheral viral-specific CD8⁺ response. Overall, by using GIFT7-derivedhypersignalling to IL7Rα/γ_(c) under conditions of age-associated thymicinsufficiency, the properties of a unique population of resident thymicprecursors capable of repopulating T lineage cells are exploited.

Sustained postnatal T cell development depends on the continuous supplyof bone marrow derived hematopoietic stem cells as resident thymocytesare deemed to possess limited self-renewing capacity. This is evident inthat mature T cells are rapidly replaced by BM sources after transientdetection of thymic output in the periphery after thymic transplants.However, this theory has been questioned recently. Allman et al showsthat early thymic progenitors (ETPs) and BM common lymphoid progenitors(CLPs) are phenotypically diverse—postulating that thymic T lineageprecursors represent a different route than BM CLPs. See Allman et al.,Nat Immunol, 2003, 4(2):168-174. Matins et al describesthymic-autonomous T cell development with normal intrathymicdifferentiation and TCR diversification in the events of defectivereceptor tyrosine kinase Kit and γ_(c) signalling localized at the BMcompartment. See Martins et al., entitled “Thymus-autonomous T celldevelopment in the absence of progenitor import.” J Exp Med. 2012. Byeliminating BM-derived competition for thymic survival niche, residentthymocytes exhibit full self-renewing capacities. Peaudecerf et aldescribes a population of thymic precursors with the capacity to persistin the complete absence of functional bone marrow supply and definedthem phenotypically as CD3⁻CD4⁻CD8⁻CD44⁺CD25^(low) IL-7R^(low) TN1-TN2cells. See Peaudecerf et al., entitled “Thymocytes may persist anddifferentiate without any input from bone marrow progenitors.” J ExpMed. 2012. Interestingly, both studies point to the importance of bonemarrow-specific IL7-IL7Rα/γ_(c) axis disruption in restoring intrathymicself-renewal when contrasting their current findings to thelong-standing dogma of marrow-dependent thymopoiesis. Thus, thecontinuous replacement of thymic progenitors by the bone marrowrepresents the competition for survival signals (i.e. IL7) in the thymicniche instead of an intrinsic hierarchy of differentiation potentialbetween bone marrow derived and resident thymic T-lineage precursors.

Studies herein demonstrate that other than eliminatingIL7-responsiveness in the bone marrow, providing an exogenoussupraphysiological level of IL7 signalling (i.e. GIFT7) also reveals theautonomy of thymic T cell development under both immune replete andinsufficient states. Systemic administration of GIFT7 drives DN1expansion (FIG. 10C), which subsequently leads to an increase in totalnumber of thymocytes as well as DP in two weeks (FIG. 10D). Thisimplicates that the immediate effect of hypertonic IL7 signalling onthymic composition is the preferential expansion of early T cellprecursors destined to persist and differentiate along the fate of Tlymphopoiesis. This is not achievable via physiological signalling ofIL7Rα/γ_(c) as previous studies have demonstrated the limited impact ofIL7 supplementation on thymic ratio under immune competent conditions.See Chu et al., Blood, 2004, 104(4):1110-1119 and Phillips et al., JImmunol, 2004, 173(8):4867-4874. The ontogeny of resident thymicprecursors was further investigated by superimposing supraphysiologicalsignalling of IL7 on age-related thymic insufficiency. The reduction ofsurvival niche in the thymus responded to GIFT7 in a dramatic fashion:hypertrophy of cortical tissue (FIG. 11A), disruption ofcortical-medullary junction (FIG. 11A), and a 4-fold increase in thespecific subset of CD44^(int) DN1 (FIG. 12B). The increase in the RNAlevel of sjTREC per splenic TCRα⁺ T cells suggests thatGIFT7-regenerated CD44^(int) DN1 undergoes normal T cell development(i.e. α rearrangement) thus contributing to peripheral T lymphoidcompartmentalization (FIG. 11C). This is especially significant in lightof the diminishment of repertoire diversity as a cause of weakened hostdefense during ageing. Indeed, the repopulation of resident thymicprecursors via GIFT7 leads to enhanced CTL response against MCMV,presumably by expanding TCR diversity at the periphery, rendering maturelymphocytes more readily available in responding to newly-encounteredantigens (FIG. 13D). In vivo data indicates that CD44^(int) DN1 subsetis most responsive to GIFT7-mediated IL7Rα/γ_(c) hypersignalling, andits expansion in frequency and absolute numbers restores thymopoiesisand reverses aged-related atrophy. However, CD44^(int) DN1 express lowerlevels of IL7Rα compared to the CD44hi subset (FIG. 12C). This seeminglyparadoxical finding can be explained by the unique binding of fusokinesto its heterodimeric receptors. GIFT fusion induces receptor clusteringand trans-activates both the α and γ_(c) chains of the heterodimericreceptor complex, which enables signaling activation even on cells withlow level of receptor expression.

There is considerable heterogeneity and discrepancy of the ontogeny ofintrathymic T cell progenitors. Here, the existence of a subset ofresident thymocytes capable of T cell neogenesis is proposed, and itsexpansion and repopulation in aged thymi is triggered by pharmacologicIL7Rα/γ_(c) hypersignalling. Their phenotype(CD4-CD8-CD44^(int)CD25-IL7Rα^(low)) resembles the TN1-TN2 cells capableof marrow-independent thymic renewal described by Peaudecerf et al.Keratinocyte growth factor (KGF), growth hormone or ghrelin reorganizethymic architecture by acting on thymic epithelial cells (TECs) withonly modest effect on lymphoid progenitors. Γc cytokine replacementtherapy seems to have more impact on peripheral T cell pool than de novoT cell production. Here, a novel thymic trophic approach is described byproviding direct mitogenic signals to T cell precursors via IL7Rα/γ_(c)axis. Based on our phosphorylation STATS data, it is proposed that GIFT7enables a hypertonic, supraphysiological IL7Rα/γ_(c) dependent signalingeven on IL7Rα^(low) cells. Hence, the use of GIFT7 or GIFT7-enhanced Tcell precursors for the treatment of a non-exclusive listing of humanthymic hypoimmune ailments with unmet clinical needs such as congenitalor HIV-mediated acquired immunodeficiency, post-ablative cytotoxicchemotherapy and ageing.

Pharmaceutical Compositions

As used herein the language “pharmaceutically acceptable excipient” isintended to include any and all carriers, solvents, diluents,excipients, adjuvants, dispersion media, coatings, antibacterial andantifungal agents, and absorption delaying agents, and the like,compatible with pharmaceutical administration.

Suitably, the pharmaceutical composition of the disclosure comprises acarrier and/or diluent appropriate for its delivering by injection to ahuman or animal organism. Such carrier and/or diluent is non-toxic atthe dosage and concentration employed. It is selected from those usuallyemployed to formulate compositions for parental administration in eitherunit dosage or multi-dose form or for direct infusion by continuous orperiodic infusion. It is typically isotonic, hypotonic or weaklyhypertonic and has a relatively low ionic strength, such as provided bysugars, polyalcohols and isotonic saline solutions. Representativeexamples include sterile water, physiological saline (e.g. sodiumchloride), bacteriostatic water, Ringer's solution, glucose orsaccharose solutions, Hank's solution, and other aqueous physiologicallybalanced salt solutions (see for example the most current edition ofRemington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott,Williams & Wilkins). The pH of the composition of the disclosure issuitably adjusted and buffered in order to be appropriate for use inhumans or animals, typically at a physiological or slightly basic pH(between about pH 8 to about pH 9, with a special preference for pH8.5). Suitable buffers include phosphate buffer (e.g. PBS), bicarbonatebuffer and/or Tris buffer. A typical composition is formulated in 1Msaccharose, 150 mM NaCl, 1 mM MgCl2, 54 mg/l Tween 80, 10 mM Tris pH8.5. Another typical composition is formulated in 10 mg/ml mannitol, 1mg/ml HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl.

The composition of the disclosure can be in various forms, e.g. in solid(e.g. powder, lyophilized form), or liquid (e.g. aqueous). In the caseof solid compositions, the typical methods of preparation are vacuumdrying and freeze-drying which yields a powder of the active agent plusany additional desired ingredient from a previously sterile-filteredsolution thereof. Such solutions can, if desired, be stored in a sterileampoule ready for reconstitution by the addition of sterile water forready injection.

Nebulized or aerosolized formulations also form part of this disclosure.Methods of intranasal administration are well known in the art,including the administration of a droplet, spray, or dry powdered formof the composition into the nasopharynx of the individual to be treatedfrom a pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer (see forexample WO 95/11664). Enteric formulations such as gastroresistantcapsules and granules for oral administration, suppositories for rectalor vaginal administration also form part of this disclosure. Fornon-parental administration, the compositions can also includeabsorption enhancers which increase the pore size of the mucosalmembrane. Such absorption enhancers include sodium deoxycholate, sodiumglycocholate, dimethyl-beta-cyclodextrin,lauroyl-1-lysophosphatidylcholine and other substances having structuralsimilarities to the phospholipid domains of the mucosal membrane.

The composition can also contain other pharmaceutically acceptableexcipients for providing desirable pharmaceutical or pharmacodynamicproperties, including for example modifying or maintaining the pH,osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution of the formulation, modifying or maintaining release orabsorption into an the human or animal organism. For example, polymerssuch as polyethylene glycol may be used to obtain desirable propertiesof solubility, stability, half-life and other pharmaceuticallyadvantageous properties (Davis et al., 1978, Enzyme Eng. 4, 169-173;Burnham et al., 1994, Am. J. Hosp. Pharm. 51, 210-218). Representativeexamples of stabilizing components include polysorbate 80, L-arginine,polyvinylpyrrolidone, trehalose, and combinations thereof. Otherstabilizing components especially suitable in plasmid-based compositionsinclude hyaluronidase (which is thought to destabilize the extracellular matrix of the host cells as described in WO 98/53853),chloroquine, protic compounds such as propylene glycol, polyethyleneglycol, glycerol, ethanol, 1-methyl L-2-pyrrolidone or derivativesthereof, aprotic compounds such as dimethylsulfoxide (DMSO),diethylsulfoxide, di-n-propylsulfoxide, dimethylsulfone, sulfolane,dimethyl-formamide, dimethylacetamide, tetramethylurea, acetonitrile(see EP 890 362), nuclease inhibitors such as actin G (WO 99/56784) andcationic salts such as magnesium (Mg²⁺) (EP 998 945) and lithium (Li⁺)(WO 01/47563) and any of their derivatives. The amount of cationic saltin the composition of the disclosure typically ranges from about 0.1 mMto about 100 mM, and still more typically from about 0.1 mM to about 10mM. Viscosity enhancing agents include sodium carboxymethylcellulose,sorbitol, and dextran. The composition can also contain substances knownin the art to promote penetration or transport across the blood barrieror membrane of a particular organ (e.g. antibody to transferrinreceptor; Friden et al., 1993, Science 259, 373-377). A gel complex ofpoly-lysine and lactose (Midoux et al., 1993, Nucleic Acid Res. 21,871-878) or poloxamer 407 (Pastore, 1994, Circulation 90, 1-517) can beused to facilitate administration in arterial cells.

The composition of the disclosure may also comprise one or moreadjuvant(s) suitable for systemic or mucosal application in humans.Representative examples of useful adjuvants include without limitationalum, mineral oil emulsion such as Freunds complete and incomplete,lipopolysaccharide or a derivative thereof (Ribi et al., 1986,Immunology and Immunopharmacology of Bacterial Endotoxins, Plenum Publ.Corp., NY, p407-419), saponins such as QS21 (Sumino et al., 1998, J.Virol. 72, 4931-4939; WO 98/56415), Escin, Digitonin, Gypsophila orChenopodium quinoa saponins. Alternatively the composition of thedisclosure may be formulated with conventional vaccine vehicles composedof chitosan or other polycationic polymers, polylactide andpolylactide-co-glycolide particles, poly-N-acetyl glucosamine-basedpolymer matrix, particles composed of polysaccharides or chemicallymodified polysaccharides, and lipid-based particles, etc. Thecomposition may also be formulated in the presence of cholesterol toform particulate structures such as liposomes.

The composition may be administered to patients in an amount effective,especially to enhance an immune response in an animal or human organism.As used herein, the term “effective amount” refers to an amountsufficient to realize a desired biological effect. For example, aneffective amount for enhancing an immune response could be that amountnecessary to cause activation of the immune system, for instanceresulting in the development of an anti-tumor response in a cancerouspatient (e.g. size reduction or regression of the tumor into which thecomposition has been injected and/or distant tumors). The appropriatedosage may vary depending upon known factors such as the pharmacodynamiccharacteristics of the particular active agent, age, health, and weightof the host organism; the condition(s) to be treated, nature and extentof symptoms, kind of concurrent treatment, frequency of treatment, theneed for prevention or therapy and/or the effect desired. The dosagewill also be calculated dependent upon the particular route ofadministration selected. Further refinement of the calculationsnecessary to determine the appropriate dosage for treatment is routinelymade by a practitioner, in the light of the relevant circumstances. Thetiter may be determined by conventional techniques. A composition basedon vector plasmids may be formulated in the form of doses of between 1μg to 100 mg, advantageously between 10 μg and 10 mg and typicallybetween 100 μg and 1 mg. A composition based on proteins may beformulated in the form of doses of between 10 ng to 100 mg. A typicaldose is from about 1 μg to about 10 mg of the therapeutic protein per kgbody weight. The administration may take place in a single dose or adose repeated one or several times after a certain time interval. In onetypical embodiment, the composition of the present disclosure isadministered by injection using conventional syringes and needles, ordevices designed for ballistic delivery of solid compositions (WO99/27961), or needleless pressure liquid jet device (U.S. Pat. No.4,596,556; U.S. Pat. No. 5,993,412).

The composition of the disclosure can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic. Inall cases, the composition must be sterile and should be fluid to theextent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi.Sterile injectable solutions can be prepared by incorporating the activeagent (e.g., a fusion protein or infectious particles) in the requiredamount with one or a combination of ingredients enumerated above,followed by filtered sterilization.

Methods of Use

The pharmaceutical composition of the disclosure may be employed inmethods for treating or preventing a variety of diseases and pathologicconditions, including genetic diseases, congenital diseases and acquireddiseases such as infectious diseases (e.g. viral and/or bacterialinfections), cancer, immune deficiency diseases, and autoimmunediseases. Accordingly, the present disclosure also encompasses the useof the fusion protein, vector, infectious viral particle, host cell orcomposition of the disclosure for the preparation of a drug intended fortreating or preventing such diseases, and especially cancer or aninfectious disease.

The composition of the present disclosure is particularly intended forthe preventive or curative treatment of disorders, conditions ordiseases associated with cancer. The term “cancer” encompasses anycancerous conditions including diffuse or localized tumors, metastasis,cancerous polyps and preneoplastic lesions (e.g. dysplasies) as well asdiseases which result from unwanted cell proliferation. A variety oftumors may be selected for treatment in accordance with the methodsdescribed herein. In general, solid tumors are typical. Cancers whichare contemplated in the context of the disclosure include withoutlimitation glioblastoma, sarcoma, melanomas, mastocytoma, carcinomas aswell as breast cancer, prostate cancer, testicular cancer, ovariancancer, endometrial cancer, cervical cancer (in particular, thoseinduced by a papilloma virus), lung cancer (e.g. lung carcinomasincluding large cell, small cell, squamous and adeno-carcinomas), renalcancer, bladder cancer, liver cancer, colon cancer, anal cancer,pancreatic cancer, stomach cancer, gastrointestinal cancer, cancer ofthe oral cavity, larynx cancer, brain and CNS cancer, skin cancer (e.g.melanoma and non-melanoma), blood cancer (lymphomas, leukemia,especially if they have developed in solid mass), bone cancer,retinoblastoma and thyroid cancer. In one typical embodiment of the useof the disclosure, the composition is administered into or in closeproximity to a solid tumor.

In certain embodiments, the disclosure contemplates uses of conjugatesdisclosed herein in autologous immune enhancement therapy (AIET). AIETis a treatment method in which immune or cancer cells, e.g.,lymphokine-activated killer (LAK) cells, natural killer (NK) cells,cytotoxic T lymphocytes (CTLs), dendritic cells (DCs), are taken outfrom the body of a subject which are cultured and processed to activatethem until their resistance to cancer is strengthened and then the cellsare put back in the body. The cells, antibodies, and organs of theimmune system work to protect and defend the body against the tumorcells. In certain embodiments, the disclosure contemplates mixingharvested cells with conjugates of GM-CSF and IL-7 to activate them. Incertain embodiments, the disclosure contemplates administeringconjugates of GM-CSF and IL-7 when the cells are administered back tothe subject.

In certain embodiments, the disclosure contemplates the administrationof sipuleucel-T (PROVENGE) in combination with a conjugate of GM-CSF andIL-7. PROVENGE consists of autologous peripheral blood mononuclearcells, including antigen presenting cells (APCs), that have beenactivated during a culture period with a recombinant human protein,PAP-GM-CSF, consisting of prostatic acid phosphatase (PAP), an antigenexpressed in prostate cancer tissue, linked to GM-CSF. In certainembodiments, the disclosure relates to a conjugate comprising PAP,GM-CSF, and IL-7, and uses in activating antigen presenting cells inperipheral blood mononuclear cells. The peripheral blood mononuclearcells of the subject may be obtained via a standard leukapheresisprocedure prior to infusion. During culture, the recombinant antigen canbind to and be processed by antigen presenting cells (APCs). Therecombinant antigen is believed to direct the immune response to PAP.The infused product is believed to contain antigen presenting cells,dendritic cells, T cells, B cells, natural killer (NK) cells, and othercells. Typically each dose contains more than 50 million autologousCD54⁺ cells activated with PAP-GM-CSF or PAP-GM-CSF-IL-7. The potency istypically evaluated by measuring the increased expression of the CD54molecule, also known as ICAM-1, on the surface of APCs after culturewith PAP-GM-CSF or PAP-CM-CSF-IL-7. CD54 is a cell surface molecule thatplays a role in the immunologic interactions between APCs and T cells,and is considered a marker of immune cell activation.

In certain embodiments, the disclosure contemplates methods for treatingcancer comprising administering any GM-CSF and IL-7 conjugate disclosedherein as an immune adjuvant in combination with a vector that encodes atumor associated antigen/cancer marker, such as PSA, PAP, and optionallyencoding other co-stimulatory molecules selected from, B7-1, B7-2,ICAM-1, GM-CSF, leukocyte function-associated antigen-3 (LFA-3). Otherembodiments contemplated for the treatment of cancer includeadministering an effective amount of a vector that encodes a GM-CSF andIL-7 conjugate disclosed herein and optionally further encodes a tumorassociated antigen/cancer marker and optionally encodes otherco-stimulatory molecules to a subject. PROSTVAC is a recombinant vectorencoding costimulatory molecules, as well as PSA as a vaccine target.Plasmid DNA is incorporated into either vaccinia or fowlpox viruses bymeans of a packing cell line. Patients are treated with a vaccinia primefollowed by a series of fowlpox-based boosts.

In certain embodiments, the disclosure relates to methods of treatingcancer comprising administering a GM-CSF and IL-7 conjugate incombination with an anti-CTLA-4 antibody. Anti-CTLA-4 antibody iscontemplated to be administered in combination with any of the methodsdisclosed herein. It is believed that it binds to CTLA-4 surfaceglycoprotein on T-cell surface, minimizing immune autoregulation andpotentially enhancing antitumor activity. Interactions between B7molecules on antigen-presenting cells and CTLA-4 on tumor-specific Tcells are inhibitory. Thus, CTLA-4 engagement negatively regulates theproliferation and function of such T cells. Under certain conditions,blocking CTLA-4 with a monoclonal antibody (ipilimumab or tremilimumab)restores T-cell function.

Other embodiments contemplated for the treatment of cancer includemethods that utilize the extraction of cancer cells from a subject andincorporate glycosyl-phosphatidylinositol (GPI)-anchored co-stimulatorymolecules such as B7-1 and B7-2 into tumor cell membranes optionallywith a conjugate GM-CSF and IL-7 anchored GPI, and administering themodified cells to the subject in combination with a conjugate of GM-CSFand IL-7 to elicit an immune response. See e.g., McHugh et al., CancerRes., 1999, 59(10):2433-7; Poloso et al., Mol Immunol., 2002,38(11):803-16; and Nagarajan & Selvaraj, Cancer Res., 2002,62(10):2869-74.

Other pathologic diseases and conditions are also contemplated in thecontext of the disclosure, especially infectious diseases associatedwith an infection by a pathogen such as fungi, bacteria, protozoa andviruses. Representative examples of viral pathogens include withoutlimitation human immunodeficiency virus (e.g. HIV-1 or HIV-2), humanherpes viruses (e.g. HSV1 or HSV2), cytomegalovirus, Rotavirus, EpsteinBarr virus (EBV), hepatitis virus (e.g. hepatitis B virus, hepatitis Avirus, hepatitis C virus and hepatitis E virus), varicella-zoster virus(VZV), paramyxoviruses, coronaviruses; respiratory syncytial virus,parainfluenza virus, measles virus, mumps virus, flaviviruses (e.g.Yellow Fever Virus, Dengue Virus, Tick-borne encephalitis virus,Japanese Encephalitis Virus), influenza virus, and typically humanpapilloma viruses (e.g. HPV-6, 11, 16, 18, 31. 33). Representativeexamples of bacterial pathogens include Neisseria (e.g. N. gonorrhea andN. meningitidis); Bordetella (e.g. B. pertussis, B. parapertussis and B.bronchiseptica), Mycobacteria (e.g. M. tuberculosis, M. bovis, M.leprae, M. avium, M. paratuberculosis, M. smegmatis); Legionella (e.g.L. pneumophila); Escherichia (e.g. enterotoxic E. coli, enterohemorragicE. coli, enteropathogenic E. coli); Vibrio (e.g. V. cholera); Shigella(e.g. S. sonnei, S. dysenteriae, S. flexnerii); Salmonella (e.g. S.typhi, S. paratyphi, S. choleraesuis, S. enteritidis); Listeria (e.g. L.monocytogenes); Helicobacter (e.g. H. pylori); Pseudomonas (e.g. P.aeruginosa); Staphylococcus (e.g. S. aureus, S. epidermidis);Enterococcus (e.g. E. faecalis, E. faecium), Clostridium (e.g. C.tetani, C. botulinum, C. difficile); Bacillus (e.g. B. anthracis);Corynebacterium (e.g. C. diphtheriae), and Chlamydia (e.g. C.trachomatis, C. pneumoniae, C. psittaci). Representative examples ofparasite pathogens include Plasmodium (e.g. P. falciparum), Toxoplasma(e.g. T. gondii) Leshmania (e.g. L. major), Pneumocystis (e.g. P.carinii), Trichomonas (e.g. T. vaginalis), Schisostoma (e.g. S.mansoni). Representative examples of fungi include Candida (e.g. C.albicans) and Aspergillus.

Examples of autoimmune diseases include, but are not limited to,multiple sclerosis (MS), scleroderma, rheumatoid arthritis, autoimmunehepatitis, diabetes mellitus, ulcerative colitis, Myasthenia gravis,systemic lupus erythematosus, Graves' disease, idiopathicthrombocytopenia purpura, hemolytic anemia, multiplemyositis/dermatomyositis, Hashimoto's disease, autoimmune hypocytosis,Sjogren's syndrome, angitis syndrome and drug-induced autoimmunediseases (e.g., drug-induced lupus).

Moreover, as mentioned above, the fusion protein, nucleic acid molecule,vector, infectious particle, host cell and/or composition of the presentdisclosure can be used as an adjuvant to enhance the immune response ofan animal or human organism to a particular antigen. This particular useof the present disclosure may be made in combination with one or moretransgenes or transgene products as defined above, e.g. for purposes ofimmunotherapy. Typically, the active agent (e.g. fusion protein,infectious particle or pharmaceutical composition of the disclosure) isadministered in combination with one or more transgenes or transgeneproducts. Accordingly, there is typically also provided a compositioncomprising in combination a transgene product (e.g. a viral antigen or asuicide gene product) and a fusion protein as well as a compositioncomprising vector(s) or viral particles encoding a transgene product anda fusion protein. The transgene and the fusion-encoding nucleic acidsequences may be expressed from the same vector or from separate vectorswhich may have the same origin (e.g. adenoviral vectors) or a differentorigin (e.g. a MVA vector encoding the particular antigen and anadenoviral vector encoding the fusion protein). The fusion protein andthe transgene product (or their respective encoding vectors) can beintroduced into the host cell or organism either concomitantly orsequentially either via the mucosal and/or systemic route.

Combination Therapies

The cancer treatments disclosed herein can be applied as a sole therapyor can involve, conventional surgery or radiotherapy, hormonal therapy,or chemotherapy. Such chemotherapy can include one or more of thefollowing categories of anti-tumour agents:

(i) antiproliferative/antineoplastic drugs and combinations thereof, asused in medical oncology, such as alkylating agents (for examplecis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan,chlorambucil, busulfan and nitrosoureas); antimetabolites (for exampleantifolates such as fluoropyrimidines like 5-fluorouracil andgemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinosideand hydroxyurea); antitumour antibiotics (for example anthracyclineslike adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin,idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitoticagents (for example vinca alkaloids like vincristine, vinblastine,vindesine and vinorelbine and taxoids like taxol and taxotere); andtopoisomerase inhibitors (for example epipodophyllotoxins like etoposideand teniposide, amsacrine, topotecan and camptothecin); and proteosomeinhibitors (for example bortezomib [Velcade®]); and the agent anegrilide[Agrylin®]; and the agent alpha-interferon

(ii) cytostatic agents such as antioestrogens (for example tamoxifen,toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptordown regulators (for example fulvestrant), antiandrogens (for examplebicalutamide, flutamide, nilutamide and cyproterone acetate), LHRHantagonists or LHRH agonists (for example goserelin, leuprorelin andbuserelin), progestogens (for example megestrol acetate), aromataseinhibitors (for example as anastrozole, letrozole, vorazole andexemestane) and inhibitors of 5α-reductase such as finasteride;

(iii) agents which inhibit cancer cell invasion (for examplemetalloproteinase inhibitors like marimastat and inhibitors of urokinaseplasminogen activator receptor function);

(iv) inhibitors of growth factor function, for example such inhibitorsinclude growth factor antibodies, growth factor receptor antibodies (forexample the anti-Her2 antibody trastuzumab and the anti-epidermal growthfactor receptor (EGFR) antibody, cetuximab), farnesyl transferaseinhibitors, tyrosine kinase inhibitors and serine/threonine kinaseinhibitors, for example inhibitors of the epidermal growth factor familyfor example EGFR family tyrosine kinase inhibitors such as:N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (gefitinib),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib), and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazolin-4-amine(CI 1033), for example inhibitors of the platelet-derived growth factorfamily and for example inhibitors of the hepatocyte growth factorfamily, for example inhibitors of phosphotidylinositol 3-kinase (PI3K)and for example inhibitors of mitogen activated protein kinase kinase(MEK1/2) and for example inhibitors of protein kinase B (PKB/Akt), forexample inhibitors of Src tyrosine kinase family and/or Abelson (AbI)tyrosine kinase family such as dasatinib (BMS-354825) and imatinibmesylate (Gleevec™); and any agents that modify STAT signalling;

(v) antiangiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, (for example the anti-vascularendothelial cell growth factor antibody bevacizumab [Avastin™]) andcompounds that work by other mechanisms (for example linomide,inhibitors of integrin ocvβ3 function and angiostatin);

(vi) vascular damaging agents such as Combretastatin A4;

(vii) antisense therapies, for example those which are directed to thetargets listed above, such as an anti-RAS antisense; and

(viii) immunotherapy approaches, including for example ex-vivo andin-vivo approaches to increase the immunogenicity of subject tumourcells, such as transfection with cytokines such as interleukin 2,interleukin 4 or granulocyte-macrophage colony stimulating factor,approaches to decrease T-cell energy, approaches using transfectedimmune cells such as cytokine-transfected dendritic cells, approachesusing cytokine-transfected tumour cell lines and approaches usinganti-idiotypic antibodies, and approaches using the immunomodulatorydrugs thalidomide and lenalidomide [Revlimid®].

The combination therapy also contemplates use of the disclosedpharmaceutical compositions with radiation therapy or surgery, as analternative, or a supplement, to a second therapeutic orchemotherapeutic agent.

A typical chronic lymphocytic leukemia (CLL) chemotherapeutic planincludes combination chemotherapy with chlorambucil or cyclophosphamide,plus a corticosteroid such as prednisone or prednisolone. The use of acorticosteroid has the additional benefit of suppressing some relatedautoimmune diseases, such as immunohemolytic anemia or immune-mediatedthrombocytopenia. In resistant cases, single-agent treatments withnucleoside drugs such as fludarabine, pentostatin, or cladribine may besuccessful. Patients may consider allogeneic or autologous bone marrowtransplantation. In certain embodiments, the disclosure contemplatescombination treatments using conjugates disclosed herein in combinationwith chloroambucil, cyclophosphamide, prednisone, prednisolone,fludarabine, pentostatin, and/or cladribine or combinations thereof.

Treatment of acute lymphoblastic leukemia typically includeschemotherapy to bring about bone marrow remission. Typical regimentsinclude prednisone, vincristine, and an anthracycline drug,L-asparaginase or cyclophosphamide. Other options include tprednisone,L-asparaginase, and vincristine. Consolidation therapy orintensification therapy to eliminate any remaining leukemia may includeantimetabolite drugs such as methotrexate and 6-mercaptopurine (6-MP).In certain embodiments, the disclosure contemplates combinationtreatments using conjugates disclosed herein in combination with COP,CHOP, R-CHOP, imatinib, alemtuzumab, vincristine, L-asparaginase orcyclophosphamide, methotrexate and/or 6-mercaptopurine (6-MP). COPrefers to a chemotherapy regimen used in the treatment of lymphoma ofcyclophosphamide, vincristine, and prednisone or prednisolone andoptionally hydroxydaunorubicin (CHOP) and optionally rituximab (R-CHOP).

In some embodiments, the disclosure relates to treating a viralinfection by administering a GM-CSF and IL-7 conjugate in combinationwith a second antiviral agent. In further embodiments, a GM-CSF and IL-7conjugate is administered in combination with one or more of thefollowing agents: abacavir, acyclovir, acyclovir, adefovir, amantadine,amprenavir, ampligen, arbidol, atazanavir, atripla, boceprevir,cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol,edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir,famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet,ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir,inosine, interferon type III, interferon type II, interferon type I,lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone,nelfinavir, nevirapine, nexavir, oseltamivir (Tamiflu), peginterferonalfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin,raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir,stavudine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine,trizivir, tromantadine, truvada, valaciclovir (Valtrex), valganciclovir,vicriviroc, vidarabine, viramidine zalcitabine, zanamivir (Relenza),and/or zidovudine (AZT).

Antiviral agents include, but are not limited to, protease inhibitors(PIs), integrase inhibitors, entry inhibitors (fusion inhibitors),maturation inhibitors, and reverse transcriptase inhibitors(anti-retrovirals). Combinations of antiviral agents create multipleobstacles to viral replication, i.e., to keep the number of offspringlow and reduce the possibility of a superior mutation. If a mutationthat conveys resistance to one of the agents being taken arises, theother agents continue to suppress reproduction of that mutation. Forexample, a single anti-retroviral agent has not been demonstrated tosuppress an HIV infection for long. These agents are typically taken incombinations in order to have a lasting effect. As a result, thestandard of care is to use combinations of anti-retrovirals.

Reverse transcribing viruses replicate using reverse transcription,i.e., the formation of DNA from an RNA template. Retroviruses oftenintegrate the DNA produced by reverse transcription into the hostgenome. They are susceptible to antiviral drugs that inhibit the reversetranscriptase enzyme. In certain embodiments the disclosure relates tomethods of treating viral infections by administering a GM-CSF and IL-7conjugate, and a retroviral agent such as nucleoside and nucleotidereverse transcriptase inhibitors (NRTI) and/or a non-nucleoside reversetranscriptase inhibitors (NNRTI). Examples of nucleoside reversetranscriptase inhibitors include zidovudine, didanosine, zalcitabine,stavudine, lamivudine, abacavir, emtricitabine, entecavir, apricitabine.Examples of nucleotide reverse transcriptase inhibitors includetenofovir and adefovir. Examples of non-nucleoside reverse transcriptaseinhibitors include efavirenz, nevirapine, delavirdine, and etravirine.

In certain embodiments, the disclosure relates to methods of treating aviral infection by administering a GM-CSF and IL-7 conjugate incombination with an antiviral drug, e.g., 2′,3′-dideoxyinosine and acytostatic agent, e.g., hydroxyurea.

Human immunoglobulin G (IgG) antibodies are believed to have opsonizingand neutralizing effects against certain viruses. IgG is sometimesadministered to a subject diagnosed with immune thrombocytopenic purpura(ITP) secondary to a viral infection since certain viruses such as, HIVand hepatitis, cause ITP. In certain embodiments, the disclosure relatesto methods of treating or preventing viral infections comprisingadministering a GM-CSF and IL-7 conjugate in combination with animmunoglobulin to a subject. IgG is typically manufactured from largepools of human plasma that are screened to reduce the risk of undesiredvirus transmission. The Fc and Fab functions of the IgG molecule areusually retained. Therapeutic IgGs include Privigen, Hizentra, andWinRho. WinRho is an immunoglobulin (IgG) fraction containing antibodiesto the Rho(D) antigen (D antigen). The antibodies have been shown toincrease platelet counts in Rho(D) positive subjects with ITP. Themechanism is thought to be due to the formation of anti-Rho(D)(anti-D)-coated RBC complexes resulting in Fc receptor blockade, thussparing antibody-coated platelets.

In some embodiments, the disclosure relates to treating a bacterialinfection by administering a GM-CSF and IL-7 conjugate in combinationwith an antibiotic drug. In further embodiments, the subject isco-administered with an antibiotic selected from the group comprising ofSulfonamides, Diaminopyrimidines, Quinolones, Beta-lactam antibiotics,Cephalosporins, Tetracyclines, Notribenzene derivatives,Aminoglycosides, Macrolide antibiotics, Polypeptide antibiotics,Nitrofuran derivatives, Nitroimidazoles, Nicotinin acid derivatives,Polyene antibiotics, Imidazole derivatives or Glycopeptide, Cycliclipopeptides, Glycylcyclines and Oxazolidinones. In further embodiments,these antibiotics include but are not limited to Sulphadiazine,Sulfones—[Dapsone (DDS) and Paraaminosalicyclic (PAS)], Sulfanilamide,Sulfamethizole, Sulfamethoxazole, Sulfapyridine, Trimethoprim,Pyrimethamine, Nalidixic acids, Norfloxacin, Ciproflaxin, Cinoxacin,Enoxacin, Gatifloxacin, Gemifloxacin, Grepafloxacin, Levofloxacin,Lomefloxacin, Moxifloxacin, Ofloxacin, Pefloxacin, Sparfloxacin,Trovafloxacin, Penicillins (Amoxicillin, Ampicillin, Azlocillin,Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Hetacillin,Oxacillin, Mezlocillin, Penicillin G, Penicillin V, Piperacillin),Cephalosporins (Cefacetrile, Cefadroxil, Cefalexin, Cefaloglycin,Cefalonium, Cefaloridin, Cefalotin, Cefapirin, Cefatrizine, Cefazaflur,Cefazedone, Cefazolin, Cefradine, Cefroxadine, Ceftezole, Cefaclor,Cefonicid, Ceforanide, Cefprozil, Cefuroxime, Cefuzonam, Cefmetazole,Cefoteta, Cefoxitin, Cefcapene, Cefdaloxime, Cefdinir, Cefditoren,Cefetamet, Cefixime, Cefmenoxime, Cefodizime, Cefoperazone, Cefotaxime,Cefotiam, Cefpimizole, Cefpiramide, Cefpodoxime, Cefteram, Ceftibuten,Ceftiofur, Ceftiolen, Ceftizoxime, Ceftriaxone, Cefoperazone,Ceftazidime, Cefepime), Moxolactam, Carbapenems (Imipenem, Ertapenem,Meropenem) Monobactams (Aztreonam)Oxytetracycline, Chlortetracycline,Clomocycline, Demeclocycline, Tetracycline, Doxycycline, Lymecycline,Meclocycline, Methacycline, Minocycline, Rolitetracycline,Chloramphenicol, Amikacin, Gentamicin, Framycetin, Kanamycin, Neomicin,Neomycin, Netilmicin, Streptomycin, Tobramycin, Azithromycin,Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin,Telithromycin, Polymyxin-B, Colistin, Bacitracin, TyrothricinNotrifurantoin, Furazolidone, Metronidazole, Tinidazole, Isoniazid,Pyrazinamide, Ethionamide, Nystatin, Amphotericin-B, Hamycin,Miconazole, Clotrimazole, Ketoconazole, Fluconazole, Rifampacin,Lincomycin, Clindamycin, Spectinomycin, Chloramphenicol, Clindamycin,Colistin, Fosfomycin, Loracarbef, Metronidazole, Nitrofurantoin,Polymyxin B, Polymyxin B Sulfate, Procain, Spectinomycin, Tinidazole,Trimethoprim, Ramoplanin, Teicoplanin, Vancomycin, Trimethoprim,Sulfamethoxazole, and/or Nitrofurantoin.

Vectors

The term “vector” as used herein refers to both expression andnon-expression vectors and includes viral as well as non-viral vectors,including autonomous self-replicating circular plasmids. Where arecombinant microorganism or cell culture is described as hosting an“expression vector,” this includes both extra-chromosomal circular DNAand DNA that has been incorporated into the host chromosome(s). Typicalvectors of the disclosure are expression vectors. An expression vectorcontains multiple genetic elements positionally and sequentiallyoriented, i.e., operatively linked with other necessary elements suchthat nucleic acid molecule in the vector encoding the fusion proteins ofthe disclosure can be transcribed, and when necessary, translated in thehost cells.

Any type of vector can be used in the context of the present disclosure,whether of plasmid or viral origin, whether it is an integrating ornon-integrating vector. Such vectors are commercially available ordescribed in the literature. Contemplated in the context of thedisclosure are vectors for use in gene therapy (i.e. which are capableof delivering the nucleic acid molecule to a target cell) as well asexpression vectors for use in recombinant techniques (i.e. which arecapable for example of expressing the nucleic acid molecule of thedisclosure in cultured host cells).

The vectors of the disclosure can function in prokaryotic or eukaryoticcells or in both (shuttle vectors). Suitable vectors include withoutlimitation vectors derived from bacterial plasmids, bacteriophages,yeast episomes, artificial chromosomes, such as BAC, PAC, YAC, or MAC,and vectors derived from viruses such as baculoviruses, papovaviruses(e.g. SV40), herpes viruses, adenoviruses, adenovirus-associated viruses(AAV), poxviruses, foamy viruses, and retroviruses. Vectors may also bederived from combinations of these sources such as those derived fromplasmid and bacteriophage genetic elements, e.g. cosmids and phagemids.Viral vectors can be replication-competent, conditionally replicative orreplication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Examples of suitable plasmids include but are not limited to thosederived from pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript(Stratagene), p Poly (Lathe et al., 1987, Gene 57, 193-201), pTrc (Amannet al., 1988, Gene 69, 301-315) and pET 11d (Studier et al., 1990, GeneExpression Technology: Methods in Enzymology 185, 60-89). It is wellknown that the four of the plasmid can affect the expression efficiency,and it is typical that a large fraction of the vector be in supercoiledform. Examples of vectors for expression in yeast (e.g. S. cerevisiae)include pYepSec1 (Baldari et al., 1987, EMBO J. 6, 229-234), pMFa (Kujanet al., 1982, Cell 30, 933-943), pJRY88 (Schultz et al., 1987, Gene 54,113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Thevectors of the disclosure can also be derived from baculoviruses to beexpressed in cultured insect cells (e.g. Sf 9 cells).

According to a typical embodiment of the disclosure, the nucleic acidmolecules described herein are expressed by using mammalian expressionvectors. Examples of mammalian expression vectors include pREP4, pCEP4(Invitrogene), pCI (Promega), pCDM8 (Seed, 1987, Nature 329, 840) andpMT2PC (Kaufman et al., 1987, EMBO J. 6, 187-195). The expressionvectors listed herein are provided by way of example only of somewell-known vectors available to those of ordinary skill in the art. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance, propagation or expression of the nucleic acidmolecules described herein.

Moreover, the vector of the present disclosure may also comprise amarker gene in order to select or to identify the transfected cells(e.g. by complementation of a cell auxotrophy or by antibioticresistance), stabilizing elements (e.g. cer sequence; Summers andSherrat, 1984, Cell 36, 1097-1103), integrative elements (e.g. LTR viralsequences and transposons) as well as elements providing aself-replicating function and enabling the vector to be stablymaintained in cells, independently of the copy number of the vector inthe cell. Markers include tetracycline or ampicillin-resistance genesfor prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective. The self-replicatingfunction may be provided by using a viral origin of replication andproviding one or more viral replication factors that are required forreplication mediated by that particular viral origin (WO 95/32299).Origins of replication and any replication factors may be obtained froma variety of viruses, including Epstein-Barr virus (EBV), human andbovine papilloma viruses and papovavirus BK.

Typical vectors of the present disclosure are viral vectors andespecially adenoviral vectors, which have a number of well-documentedadvantages as vectors for gene therapy. The adenoviral genome consistsof a linear double-stranded DNA molecule of approximately 36 kb carryingmore than about thirty genes necessary to complete the viral cycle. Theearly genes are divided into 4 regions (E1 to E4) that are essential forviral replication (Pettersson and Roberts, 1986, In Cancer Cells (Vol4): DNA Tumor Viruses, Botchan and Glodzicker Sharp Eds pp 37-47, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.; Halbert et al.,1985, J. Virol. 56, 250-257) with the exception of the E3 region, whichis believed dispensable for viral replication based on the observationthat naturally-occurring mutants or hybrid viruses deleted within the E3region still replicate like wild-type viruses in cultured cells (Kellyand Lewis, 1973, J. Virol. 12, 643-652). The E1 gene products encodeproteins responsible for the regulation of transcription of the viralgenome. The E2 gene products are required for initiation and chainelongation in viral DNA synthesis. The proteins encoded by the E3prevent cytolysis by cytotoxic T cells and tumor necrosis factor (Woldand Gooding, 1991, Virology 184, 1-8). The proteins encoded by the E4region are involved in DNA replication, late gene expression andsplicing and host cell shut off (Halbert et al., 1985, J. Virol. 56,250-257). The late genes (L1 to L5) encode in their majority thestructural proteins constituting the viral capsid. They overlap at leastin part with the early transcription units and are transcribed from aunique promoter (MLP for Major Late Promoter). In addition, theadenoviral genome carries at both extremities cis-acting 5′ and 3′ ITRs(Inverted Terminal Repeat) and the encapsidation region, both essentialfor DNA replication. The ITRs harbor origins of DNA replication whereasthe encapsidation region is required for the packaging of adenoviral DNAinto infectious particles.

The adenoviral vectors for use in accordance with the presentdisclosure, typically infects mamailain cells. It can be derived fromany human or animal source, in particular canine (e.g. CAV-1 or CAV-2;Genbank ref CAV1GENOM and CAV77082 respectively), avian (Genbank refAAVEDSDNA), bovine (such as BAV3; Seshidhar Reddy et al., 1998, J.Virol. 72, 1394-1402), murine (Genbank ref ADRMUSMAV1), ovine, feline,porcine or simian adenovirus or alternatively from a hybrid thereof. Anyserotype can be employed from the adenovirus serotypes 1 through 51. Forinstance, an adenovirus can be of subgroup A (e.g. serotypes 12, 18, and31), subgroup B (e.g. serotypes 3, 7, 11, 14, 16, 21, 34, and 35),subgroup C (e.g. serotypes 1, 2, 5, and 6), subgroup D (e.g. serotypes8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroupE (serotype 4), subgroup F (serotypes 40 and 41), or any otheradenoviral serotype. However, the human adenoviruses of the B or Csub-group are typical and especially adenoviruses 2 (Ad2), 5 (Ad5) and35 (Ad35). Generally speaking, adenoviral stocks that can be employed asa source of the cited adenovirus are currently available from theAmerican Type Culture Collection (ATCC, Rockville, Md.), or from anyother source. Moreover, such adenoviruses have been the subject ofnumerous publications describing their sequence, organization andbiology, allowing the artisan to apply them. Adenoviral vectors, methodsof producing adenoviral vectors, and methods of using adenoviral vectorsare disclosed, for example in U.S. Pat. No. 6,133,028, U.S. Pat. No.6,040,174, U.S. Pat. No. 6,110,735, U.S. Pat. No. 6,399,587, WO 00/50573and EP 1016711 for group C adenoviral vectors and for example in U.S.Pat. No. 6,492,169 and WO 02/40665 for non-group C adenoviral vectors.

In certain embodiments, the adenoviral vector of the present disclosureis replication-competent. The term “replication-competent” as usedherein refers to an adenoviral vector capable of replicating in a hostcell in the absence of any trans-complementation. In the context of thepresent disclosure, this term also encompasses replication-selective orconditionally-replicative adenoviral vectors which are engineered toreplicate better or selectively in cancer or hyperproliferative hostcells. Examples of such replication-competent adenoviral vectors arewell known in the art and readily available to those skill in the art(see, for example, Hernandez-Alcoceba et al., 2000, Human Gene Ther. 11,2009-2024; Nemunaitis et al., 2001, Gene Ther. 8, 746-759; Alemany etal., 2000, Nature Biotechnology 18, 723-727).

Replication-competent adenoviral vectors according to the disclosure canbe a wild-type adenovirus genome or can be derived therefrom byintroducing modifications into the viral genome, e.g., for the purposeof generating a conditionally-replicative adenoviral vector. Suchmodification(s) include the deletion, insertion and/or mutation of oneor more nucleotide(s) in the coding sequences and/or the regulatorysequences. Typical modifications are those that render saidreplication-competent adenoviral vector dependent on cellular activitiesspecifically present in a tumor or cancerous cell. In this regard, viralgene(s) that become dispensable in tumor cells, such as the genesresponsible for activating the cell cycle through p53 or Rb binding, canbe completely or partially deleted or mutated. By way of illustration,such conditionally-replicative adenoviral vectors can be engineered bythe complete deletion of the adenoviral MB gene encoding the 55 kDaprotein or the complete deletion of the MB region to abrogate p53binding (see for example U.S. Pat. No. 5,801,029 and U.S. Pat. No.5,846,945). This prevents the virus from inactivating tumor suppressionin normal cells, which means that the virus cannot replicate. However,the virus will replicate and lyse cells that have shut off p53 or Rbexpression through oncogenic transformation. As another example, thecomplete deletion of the E1A region makes the adenoviral vectordependent on intrinsic or IL-6-induced E1A-like activities. Optionally,an inactivating mutation may also be introduced in the E1A region toabrogate binding to the Rb. Rb defective mutation/deletion is typicallyintroduced within the E1A CR1 and/or CR2 domain (see for exampleWO00/24408). In a second strategy optionally or in combination to thefirst approach, native viral promoters controlling transcription of theviral genes can be replaced with tissue or tumor-specific promoters. Byway of illustration, regulation of the E1A and/or the E1B genes can beplaced under the control of a tumor-specific promoter such as the PSA,the kallikrein, the probasin, the AFP, the a-fetoprotein or thetelomerase reverse transcriptase (TERT) promoter (see for example U.S.Pat. No. 5,998,205, WO 99/25860, U.S. Pat. No. 5,698,443 and WO00/46355) or a cell-cycle specific promoter such as E2F-1 promoter(WO00/15820 and WO01/36650). Typical in this context is the exemplaryvector designated ONYX-411 which combines a Rb defective deletion of 8amino acid residues within the MA CR2 domain and the use of E2F-1promoter to control expression of both the E1A and E4 viral genes.

In certain embodiments, the adenoviral vector of the disclosure isreplication-defective. Replication-defective adenoviral vectors areknown in the art and can be defined as being deficient in one or moreregions of the adenoviral genome that are essential to the viralreplication (e.g., E1, E2 or E4 or combination thereof), and thus unableto propagate in the absence of trans-complementation (e.g., provided byeither complementing cells or a helper virus). The replication-defectivephenotype is obtained by introducing modifications in the viral genometo abrogate the function of one or more viral gene(s) essential to theviral replication. Typical replication-defective vectors are E1-deleted,and thus defective in E1 function. Such E1-deleted adenoviral vectorsinclude those described in U.S. Pat. No. 6,063,622; U.S. Pat. No.6,093,567; WO 94/28152; WO 98/55639 and EP 974 668 (the disclosures ofall of these publications are hereby incorporated herein by reference).A typical E1 deletion covers approximately the nucleotides (nt) 459 to3328 or 459 to 3510, by reference to the sequence of the humanadenovirus type 5 (disclosed in the Genbank under the accession number M73260 and in Chroboczek et al., 1992, Virol. 186, 280-285).

Furthermore, the adenoviral backbone of the vector may comprisemodifications (e.g. deletions, insertions or mutations) in additionalviral region(s), to abolish the residual synthesis of the viral antigensand/or to improve long-term expression of the nucleic acid molecules inthe transduced cells (see for example WO 94/28152; Lusky et al., 1998,J. Virol 72, 2022-2032; Yeh et al., 1997, FASEB J. 11, 615-623). In thiscontext, the present disclosure contemplates the use of adenoviralvectors lacking E1, or E1 and E2, or E1 and E3, or E1 and E4, or E1 andE2 and E3, or E1 and E2 and E4, or E1 and E3 and E4, or E1 and E2 and E3and E4 functions. An adenoviral vector defective for E2 function may bedeleted of all or part of the E2 region (typically within the E2A oralternatively within the E2B or within both the E2A and the E2B regions)or comprises one or more mutations, such as the thermosensitive mutationof the DBP (DNA Binding Protein) encoding gene (Ensinger et al., J.Virol. 10 (1972), 328-339). The adenoviral vector may also be deleted ofall or part of the E4 region (see, for example, EP 974 668 and WO00/12741). An exemplary E4 deletion covers approximately the nucleotidesfrom position 32994 to position 34998, by reference to the sequence ofthe human adenovirus type 5. In addition, deletions within thenon-essential E3 region (e.g. from Ad5 position 28597 to position 30469)may increase the cloning capacity, but it may be advantageous to retainthe E3 sequences coding for gp19k, 14.7K and/or RID allowing to escapethe host immune system (Gooding et al., 1990, Critical Review ofImmunology 10, 53-71) and inflammatory reactions (EP 00 440 267.3). Itis also conceivable to employ a minimal (or gutless) adenoviral vectorwhich lacks all functional genes including early (E1, E2, E3 and E4) andlate genes (L1, L2, L3, L4 and L5) with the exception of cis-actingsequences (see for example Kovesdi et al., 1997, Current Opinion inBiotechnology 8, 583-589; Yeh and Perricaudet, 1997, FASEB 11, 615-623;WO 94/12649; and WO 94/28152). The replication-deficient adenoviralvector may be readily engineered by one skilled in the art, taking intoconsideration the required minimum sequences, and is not limited tothese exemplary embodiments.

The nucleic acid molecule of the present disclosure can be inserted inany location of the adenoviral genome, with the exception of thecis-acting sequences. Typically, it is inserted in replacement of adeleted region (E1, E3 and/or E4), with a special preference for thedeleted E1 region. In addition, the expression cassette may bepositioned in sense or antisense orientation relative to the naturaltranscriptional direction of the region in question.

A retroviral vector is also suitable in the context of the presentdisclosure. Retroviruses are a class of integrative viruses whichreplicate using a virus-encoded reverse transcriptase, to replicate theviral RNA genome into double stranded DNA which is integrated intochromosomal DNA of the infected cells. The numerous vectors described inthe literature may be used within the framework of the presentdisclosure and especially those derived from murine leukemia viruses,especially Moloney (Gilboa et al., 1988, Adv. Exp. Med. Biol. 241, 29)or Friend's FB29 strains (WO 95/01447). Generally, a retroviral vectoris deleted of all or part of the viral genes gag, pol and env andretains 5′ and 3′ LTRs and an encapsidation sequence. These elements maybe modified to increase expression level or stability of the retroviralvector. Such modifications include the replacement of the retroviralencapsidation sequence by one of a retrotransposon such as VL30 (U.S.Pat. No. 5,747,323). The nucleic acid molecule of the disclosure can beinserted downstream of the encapsidation sequence, typically in oppositedirection relative to the retroviral genome.

A poxviral vector is also suitable in the context of the presentdisclosure. Poxviruses are a group of complex enveloped viruses thatdistinguish from the above-mentioned viruses by their large DNA genomeand their cytoplasmic site of replication. The genome of several membersof poxyiridae has been mapped and sequenced. It is a double-stranded DNAof approximately 200 kb coding for about 200 proteins of whichapproximately 100 are involved in virus assembly. In the context of thepresent disclosure, a poxyiral vector may be obtained from any member ofthe poxyiridae, in particular canarypox, fowlpox and vaccinia virus, thelatter being typical. Suitable vaccinia viruses include withoutlimitation the Copenhagen strain (Goebel et al., 1990, Virol. 179,247-266 and 517-563; Johnson et al., 1993, Virol. 196, 381-401), theWyeth strain and the modified Ankara (MVA) strain (Antoine et al., 1998,Virol. 244, 365-396). The general conditions for constructing poxviruscomprising a nucleic acid molecule are well known in the art (see forexample EP 83 286; EP 206 920 for Copenhagen vaccinia viruses and Mayret al., 1975, Infection 3, 6-14; Sutter and Moss, 1992, Proc. Natl.Acad. Sci. USA 89, 10847-10851, U.S. Pat. No. 6,440,422 for MVAviruses). The nucleic acid molecule of the present disclosure istypically inserted within the poxyiral genome in a non-essential locus,such as non-coding intergenic regions or any gene for which inactivationor deletion does not significantly impair viral growth and replication.Thymidine kinase gene is particularly appropriate for insertion inCopenhagen vaccinia viruses (Hruby et al., 1983, Proc. Natl. Acad. Sci.USA 80, 3411-3415; Weir et al., 1983, J. Virol. 46, 530-537). As far asMVA is concerned, insertion of the nucleic acid molecule can beperformed in any of the excisions I to VII, and typically in excision Hor III (Meyer et al., 1991, J. Gen. Virol. 72, 1031-1038; Sutter et al.,1994, Vaccine 12, 1032-1040) or in D4R locus. For fowlpox virus,although insertion within the thymidine kinase gene may be considered,the nucleic acid molecule is typically introduced into a non-codingintergenic region (see for example EP 314 569 and U.S. Pat. No.5,180,675). One may also envisage insertion in an essential viral locusprovided that the defective function be supplied in trans, via a helpervirus or by expression in the producer cell line. Suitable poxyiralvectors can be readily generated from wild type poxviruses available inrecognized collections such as ATCC (fowlpox ATCC VR-251, monkey poxATCC VR-267, swine pox ATCC VR-363, canarypox ATCC VR-111, cowpox ATCCVR-302) or ICTV (Canberra, Australia) (Copenhagen virus code58.1.1.0.001; GenBank accession number M35027).

In certain embodiments, the vectors of the disclosure comprise thenucleic acid molecule of the disclosure in a form suitable for itsexpression in a host cell or organism, which means that the nucleic acidmolecule is placed under the control of one or more regulatorysequences, selected on the basis of the vector type and/or host cell,which is operatively linked to the nucleic acid molecule to beexpressed. As used herein, the term “regulatory sequence” refers to anysequence that allows, contributes or modulates the functional regulationof the nucleic acid molecule, including replication, duplication,transcription, splicing, translation, stability and/or transport of thenucleic acid or one of its derivative (i.e. mRNA) into the host cell ororganism. In the context of the disclosure, this term encompassespromoters, enhancers and other expression control elements (e.g.,polyadenylation signals and elements that affect mRNA stability).“Operably linked” is intended to mean that the nucleic acid molecule ofinterest is linked to the regulatory sequence(s) in a manner whichallows for expression of the nucleic acid molecule (e.g., in a host cellor organism). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expression ofprotein desired, etc.

Regulatory sequences include promoters which direct constitutiveexpression of a nucleic acid molecule in many types of host cell andthose which direct expression of the nucleotide sequence only in certainhost cells (e.g., tissue-specific regulatory sequences) or in responseto specific events or exogenous factors (e.g. by temperature, nutrientadditive, hormone or other ligand).

Suitable regulatory sequences useful in the context of the presentdisclosure include, but are not limited to, the left promoter frombacteriophage lambda, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the cytomegalovirus (CMV) immediateearly promoter or enhancer (Boshart et al., 1985, Cell 41, 521-530), theadenovirus early and late promoters, the phosphoglycero kinase (PGK)promoter (Hitzeman et al., 1983, Science 219, 620-625; Adra et al.,1987, Gene 60, 65-74), the thymidine kinase (TK) promoter of herpessimplex virus (HSV)-1 and retroviral long-terminal repeats (e.g. MoMuLVand Rous sarcoma virus (RSV) LTRs). Suitable promoters useful to driveexpression of the nucleic acid molecule of the disclosure in a poxyiralvector include the 7.5K, H5R, TK, p28, p11 or K1L promoters of vacciniavirus. Alternatively, one may use a synthetic promoter such as thosedescribed in Chakrabarti et al. (1997, Biotechniques 23, 1094-1097),Hammond et al. (1997, J. Virological Methods 66, 135-138) and Kumar andBoyle (1990, Virology 179, 151-158) as well as chimeric promotersbetween early and late poxyiral promoters.

Inducible promoters are regulated by exogenously supplied compounds, andinclude, without limitation, the zinc-inducible metallothionein (MT)promoter (Mc Ivor et al., 1987, Mol. Cell. Biol. 7, 838-848), thedexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter,the T7 polymerase promoter system (WO 98/10088), the ecdysone insectpromoter (No et al., 1996, Proc. Natl. Acad. Sci. USA 93, 3346-3351),the tetracycline-repressible promoter (Gossen et al., 1992, Proc. Natl.Acad. Sci. USA 89, 5547-5551), the tetracycline-inducible promoter (Kimet al., 1995, J. Virol. 69, 2565-2573), the RU486-inducible promoter(Wang et al., 1997, Nat. Biotech. 15, 239-243 and Wang et al., 1997,Gene Ther. 4, 432-441) and the rapamycin-inducible promoter (Magari etal., 1997, J. Clin. Invest. 100, 2865-2872).

The regulatory sequences in use in the context of the present disclosurecan also be tissue-specific to drive expression of the nucleic acidmolecule in the tissues where therapeutic benefit is desired. Exemplaryliver-specific regulatory sequences include but are not limited to thoseof HMG-CoA reductase (Luskey, 1987, Mol. Cell. Biol. 7, 1881-1893);sterol regulatory element 1 (SRE-1; Smith et al., 1990, J. Biol. Chem.265, 2306-2310); albumin (Pinkert et al., 1987, Genes Dev. 1, 268-277);phosphoenol pyruvate carboxy kinase (PEPCK) (Eisenberger et al., 1992,Mol. Cell. Biol. 12, 1396-1403); human C-reactive protein (CRP) (Li etal., 1990, J. Biol. Chem. 265, 4136-4142); human glucokinase (Tanizawaet al., 1992, Mol. Endocrinology. 6, 1070-1081); cholesterol 7-alphahydroylase (CYP-7) (Lee et al., 1994, J. Biol. Chem. 269, 14681-14689);alpha-1 antitrypsin (Ciliberto et al., 1985, Cell 41, 531-540);insulin-like growth factor binding protein (IGFBP-1) (Babajko et al.,1993, Biochem Biophys. Res. Comm. 196, 480-486); human transferrin(Mendelzon et al., 1990, Nucl. Acids Res. 18, 5717-5721); collagen typeI (Houglum et al., 1994, J. Clin. Invest. 94, 808-814) and FIX (U.S.Pat. No. 5,814,716) genes. Exemplary prostate-specific regulatorysequences include but are not limited to those of the prostatic acidphosphatase (PAP) (Balms et al., 1994, Biochim. Biophys. Acta. 1217,188-194); prostatic secretory protein 94 (PSP 94) (Nolet et al., 1991,Biochim. Biophys. Acta 1089, 247-249); prostate specific antigen complex(Kasper et al., 1993, J. Steroid Biochem. Mol. Biol. 47, 127-135); humanglandular kallikrein (hgt-1) (Lilja et al., 1993, World J. Urology 11,188-191) genes. Exemplary pancreas-specific regulatory sequences includebut are not limited to those of pancreatitis associated protein promoter(Dusetti et al., 1993, J. Biol. Chem. 268, 14470-14475); elastase 1transcriptional enhancer (Kruse et al., 1993, Genes and Development 7,774-786); pancreas specific amylase and elastase enhancer/promoter (Wuet al., 1991, Mol. Cell. Biol. 11, 4423-4430; Keller et al., 1990, Genes& Dev. 4, 1316-1321); pancreatic cholesterol esterase gene promoter(Fontaine et al., 1991, Biochemistry 30, 7008-7014) and the insulin genepromoter (Edlund et al., 1985, Science 230, 912-916). Exemplaryneuron-specific regulatory sequences include but are not limited toneuron-specific enolase (NSE) (Forss-Petter et al., 1990, Neuron 5,187-197) and the neurofilament (Byrne and Ruddle, 1989, Proc. Natl.Acad. Sci. USA 86, 5473-5477) gene promoters. Exemplary regulatorysequences for expression in the brain include but are not limited to theneurofilament heavy chain (NF-H) promoter (Schwartz et al., 1994, J.Biol. Chem. 269, 13444-13450). Exemplary lymphoid-specific regulatorysequences include but are not limited to the human CGL1/granzyme Bpromoter (Hanson et al., 1991, J. Biol. Chem. 266, 24433-24438);terminal deoxy transferase (TdT), lymphocyte specific tyrosine proteinkinase (p561ck) promoters (Lo et al., 1991, Mol. Cell. Biol. 11,5229-5243); the human CD2 promoter/enhancer (Lake et al., 1990, EMBO J.9, 3129-3136), the human NK and T cell specific activation (NKG5)(Houchins et al., 1993, Immunogenetics 37, 102-107), T cell receptor(Winoto and Baltimore, 1989, EMBO J. 8, 729-733) and immunoglobulin(Banerji et al., 1983, Cell 33, 729-740; Queen and Baltimore, 1983, Cell33, 741-748) promoters. Exemplary colon-specific regulatory sequencesinclude but are not limited to pp 60c-src tyrosine kinase (Talamonti etal., 1993, J. Clin. Invest 91, 53-60); organ-specific neoantigens(OSNs), mw 40 kDa (p40) (Ilantzis et al., 1993, Microbiol. Immunol. 37,119-128); and colon specific antigen-P promoter (Sharkey et al., 1994,Cancer 73, 864-877) promoters. Exemplary regulatory sequences forexpression in mammary gland and breast cells include but are not limitedto the human alpha-lactalbumin (Thean et al., 1990, British J. Cancer.61, 773-775) and milk whey (U.S. Pat. No. 4,873,316) promoters.Exemplary muscle-specific regulatory sequences include but are notlimited to SM22 (WO 98/15575; WO 97/35974), the desmin (WO 96/26284),mitochondrial creatine kinase (MCK) promoters, and the chimeric promoterdisclosed in EP 1310561. Exemplary lung-specific regulatory sequencesinclude but are not limited to the CFTR and surfactant promoters.

Additional promoters suitable for use in this disclosure can be takenfrom genes that are preferentially expressed in proliferative tumorcells. Such genes can be identified for example by display andcomparative genomic hybridization (see for example U.S. Pat. Nos.5,759,776 and 5,776,683). Exemplary tumor specific promoters include butare not limited to the promoters of the MUC-1 gene overexpressed inbreast and prostate cancers (Chen et al., 1995, J. Clin. Invest. 96,2775-2782), of the Carcinoma Embryonic Antigen (CEA)-encoding geneoverexpressed in colon cancers (Schrewe et al., 1990, Mol. Cell. Biol.10, 2738-2748), of the ERB-2 encoding gene overexpressed in breast andpancreas cancers (Harris et al., 1994, Gene Therapy 1, 170-175), of thealpha-foetoprotein gene overexpressed in liver cancers (Kanai et al.,1997, Cancer Res. 57, 461-465), of the telomerase reverse transcriptase(TERT) (WO99/27113, WO 02/053760 and Horikawa et al., 1999, Cancer Res.59, 826), hypoxia-responsive element (HRE), autocrine motility factorreceptor, L plasmin and hexokinase II.

Those skilled in the art will appreciate that the regulatory elementscontrolling the expression of the nucleic acid molecule of thedisclosure may further comprise additional elements for properinitiation, regulation and/or termination of transcription andtranslation into the host cell or organism. Such additional elementsinclude but are not limited to non-coding exon/intron sequences,transport sequences, secretion signal sequences, nuclear localizationsignal sequences, IRES, polyA transcription termination sequences,tripartite leader sequences, sequences involved in replication orintegration. Illustrative examples of introns suitable in the context ofthe disclosure include those isolated from the genes encoding alpha orbeta globin (i.e. the second intron of the rabbit beta globin gene;Green et al., 1988, Nucleic Acids Res. 16, 369; Karasuyama et al., 1988,Eur. J. Immunol. 18, 97-104), ovalbumin, apolipoprotein, immunoglobulin,factor IX, and factor VIII, the SV40 16S/19S intron (Okayma and Berg,1983, Mol. Cell. Biol. 3, 280-289) as well as synthetic introns such asthe intron present in the pCI vector (Promega Corp, pCI mammalianexpression vector E1731) made of the human beta globin donor fused tothe mouse immunoglobin. Where secretion of the fusion protein isdesired, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the fusion protein orheterologous to both entities involved in the fusion protein. The personof ordinary skill in the art would be aware of the numerous regulatorysequences that are useful in expression vectors.

In addition, the vector of the disclosure can further comprise one ormore transgenes (i.e. a gene of interest to be expressed together withthe nucleic acid molecule of the disclosure in a host cell or organism).Desirably, the expression of the transgene has a therapeutic orprotective activity to the disease or illness condition for which thevector of the present disclosure is being given. Suitable transgenesinclude without limitation genes encoding (i) tumor proliferationinhibitors and/or (ii) at least one specific antigen against which animmune response is desired. In a typical form of the present disclosure,the transgene product and the fusion protein act synergistically in theinduction of immune responses or in providing a therapeutic (e.g.antitumoral) benefit. Accordingly, such combinations are not onlysuitable for immunoprophylaxis of diseases, but surprisingly also forimmunotherapy of diseases such as viral, bacterial or parasiticinfections, and also chronic disorders such as cancers.

Tumor proliferation inhibitors act by directly inhibiting cell growth,or killing the tumor cells. Representative examples of tumorproliferation inhibitors include toxins and suicide genes.Representative examples of toxins include without limitation ricin (Lambet al., 1985, Eur. J. Biochem. 148, 265-270), diphtheria toxin (Twetenet al., 1985, J. Biol. Chem. 260, 10392-10394), cholera toxin (Mekalanoset al., 1983, Nature 306, 551-557; Sanchez and Holmgren, 1989, Proc.Natl. Acad. Sci. USA 86, 481-485), gelonin (Stirpe et al., 1980, J.Biol. Chem. 255, 6947-6953), antiviral protein (Barbieri et al., 1982,Biochem. J. 203, 55-59; Irvin et al., 1980, Arch. Biochem. Biophys. 200,418-425), tritin, Shigella toxin (Calderwood et al., 1987, Proc. Natl.Acad. Sci. USA 84, 4364-4368; Jackson et al., 1987, Microb. Path. 2,147-153) and Pseudomonas exotoxin A (Carroll and Collier, 1987, J. Biol.Chem. 262, 8707-8711).

Specific antigens are typically those susceptible to confer an immuneresponse, specific and/or nonspecific, antibody and/or cell-mediated,against a given pathogen (virus, bacterium, fungus or parasite) oragainst a non-self antigen (e.g. a tumor-associated antigen). Typically,the selected antigen comprises an epitope that binds to, and ispresented onto the cell surface by MHC class I proteins. Representativeexamples of specific antigens include without limitation: antigen(s) ofthe Hepatitis B surface antigen are well known in the art and include,inter alia, those PreS1, Pars2 S antigens set forth described inEuropean Patent applications EP 414 374; EP 304 578, and EP 198 474.Antigens of the Hepatitis C virus including any immunogenic antigen orfragment thereof selected from the group consisting of the Core (C), theenvelope glycoprotein E1, E2, the non-structural polypeptide NS2, NS3,NS4 (NS4a and/or NS4b), NS5 (NS5a and/or NS5b) or any combinationthereof (e.g. NS3 and NS4, NS3 and NS4 and NS5b) Antigen(s) of the HIV-1virus, especially gp120 and gp160 (as described WO 87/06260). Antigen(s)derived from the Human Papilloma Virus (HPV) considered to be associatedwith genital warts (HPV 6 or HPV 11 and others), and cervical cancer(HPV16, HPV18, HPV 31, HPV-33 and others). Contemplated HPV antigens areselected among the group consisting of E5, E6, E7, L1, and L2 eitherindividually or in combination (see for example WO 94/00152, WO94/20137, WO 93/02184, WO 90/10459, and WO 92/16636). Contemplated inthe context of the disclosure are membrane anchored forms ofnon-oncogenic variants of the early HPV-16 E6 and/or E7 antigens (asdescribed in WO 99/03885) that are particularly suitable to achieve ananti-tumoral effect against an HPV-associated cancer. Antigens fromparasites that cause malaria. For example, typical antigens fromPlasmodia falciparum include RTS (WO 93/10152), and TRAP (WO 90/01496).Other plasmodia antigens that are likely candidates are P. falciparum.MSP1, AMA1, MSP3, EBA, GLURP, RAPT, RAP2, Sequestrin, PfEMP1, Pf332,LSA1, LSA3, STARP, SALSA, PfEXP1, Pfs25, Pfs28, PFS27125, Pfs16,Pfs48/45, Pfs230 and their analogues in other Plasmodium species.

Other suitable antigens include tumour-associated antigens such as thoseassociated with prostrate, breast, colorectal, lung, pancreatic, renal,liver, bladder, sarcoma or melanoma cancers. Exemplary antigens includeMAGE 1, 3 and MAGE 4 or other MAGE antigens (WO 99/40188), PRAME, BAGE,Lage (also known as NY Eos 1) SAGE and HAGE (WO 99/53061) or GAGE(Robbins and Kawakami, 1996. Current Opinions in Immunol. 8, pps628-636). Other suitable tumor-associated antigens include those knownas prostase, including Prostate specific antigen (PSA), PAP, PSCA, PSMA.Prostase nucleotide sequence and deduced polypeptide sequence andhomologs are disclosed in Ferguson, et al. (1999, Proc. Natl. Acad. Sci.USA. 96, 3114-3119) and WO 98/12302 WO 98/20117 and WO 00/04149. Othersuitable tumour-associated antigens include those associated with breastcancer, such as BRCA-1, BRCA-2 and MUC-1 (see for example WO 92/07000).

The transgene in use in the present disclosure is placed under thecontrol of appropriate regulatory elements to permit its expression inthe selected host cell or organism in either a constitutive or induciblefashion. The choice of such regulatory elements is within the reach ofthe skilled artisan. It is typically selected from the group consistingof constitutive, inducible, tumor-specific and tissue-specific promotersas described above in connection with the expression of the fusionprotein of the present disclosure. In one example, the transgene isplaced under control of the CMV promoter to ensure high levelexpression.

The transgene in use in the present disclosure can be inserted in anylocation of the vector. According to one alternative, it is placedtypically not in close proximity of the nucleic acid molecule of thedisclosure. According to another alternative it can be placed inantisense orientation with respect to the nucleic acid molecule, inorder to avoid transcriptional interference between the two expressioncassettes. For example, in an adenoviral genome, the transgene can beinserted in a different deleted region with respect to the nucleic acidmolecule of the disclosure (E1, E3 and/or E4) or in the same deletedregion as said nucleic acid molecule but in antisense orientation to oneanother.

Introducing the nucleic acid molecule of the disclosure into a vectorbackbone can proceed by any genetic engineering strategy appropriate inthe art for any kind of vectors such as by methods described in Sambrooket al. (2001, Molecular Cloning—A Laboratory Manual, Cold Spring HarborLaboratory). Typically, for the introduction of the nucleic acidmolecule into an adenoviral vector, a bacterial plasmid comprising thefusion-encoding nucleic acid molecule is engineered to replace anadenoviral gene required for replication or assembly (e.g. E1) with thesubstitute nucleic acid molecule. The plasmid is then used as a shuttlevector, and combined with a second plasmid containing the complementaryportion of the adenovirus genome, permitting homologous recombination tooccur by virtue of overlapping adenovirus sequences in the two plasmids.The recombination can be done directly in a suitable mammalian host(such as 293 as described in Graham and Prevect, 1991, Methods inMolecular Biology, Vol 7 “Gene Transfer and Expression Protocols”; Ed E.J. Murray, The Human Press Inc, Clinton, N.J.), or else in yeast YACclones or E. coli (as described in WO 96/17070). The completedadenovirus genome is subsequently transfected into mammalian host cellsfor replication and viral encapsidation.

The present disclosure also encompasses vectors of the disclosure orparticles thereof that have been modified to allow preferentialtargeting of a particular target cell. A characteristic feature oftargeted vectors/particles of the disclosure (of both viral andnon-viral origins, such as polymer- and lipid-complexed vectors) is thepresence at their surface of a targeting moiety capable of recognizingand binding to a cellular and surface-exposed component. Such targetingmoieties include without limitation chemical conjugates, lipids,glycolipids, hormones, sugars, polymers (e.g. PEG, polylysine, PEI andthe like), peptides, polypeptides (for example JTS1 as described in WO94/40958), oligonucleotides, vitamins, antigens, lectins, antibodies andfragments thereof. They are typically capable of recognizing and bindingto cell-specific markers, tissue-specific markers, cellular receptors,viral antigens, antigenic epitopes or tumor-associated markers. In thisregard, cell targeting of adenoviruses can be carried out by geneticmodification of the viral gene encoding the capsid polypeptide presenton the surface of the virus (e.g. fiber, penton and/or pIX). Examples ofsuch modifications are described in literature (for example in Wickam etal., 1997, J. Viral. 71, 8221-8229; Amberg et al., 1997, Virol. 227,239-244; Michael et al., 1995, Gene Therapy 2, 660-668; WO 94/10323, EP02 360204 and WO 02/96939). To illustrate, inserting a sequence codingfor EGF within the sequence encoding the adenoviral fiber will allow totarget EGF receptor expressing cells. The modification of poxyiraltropism can also be achieved as described in EP 1 146 125. Other methodsfor cell specific targeting can be achieved by the chemical conjugationof targeting moieties at the surface of a viral particle.

In certain embodiments, the present disclosure relates to infectiousviral particles comprising the above-described nucleic acid molecules orvectors of the present disclosure.

The disclosure also relates to a process for producing an infectiousviral particle, comprising the steps of: (a) introducing the viralvector of the disclosure into a suitable cell line, (b) culturing saidcell line under suitable conditions so as to allow the production ofsaid infectious viral particle, and (c) recovering the producedinfectious viral particle from the culture of said cell line, and (d)optionally purifying said recovered infectious viral particle.

The vector containing the nucleic acid molecule of the disclosure can beintroduced into an appropriate cell line for propagation or expressionusing well-known techniques readily available to the person of ordinaryskill in the art. These include, but are not limited to, microinjectionof minute amounts of DNA into the nucleus of a cell (Capechi et al.,1980, Cell 22, 479-488), CaPO.sub.4-mediated transfection (Chen andOkayama, 1987, Mol. Cell Biol. 7, 2745-2752), DEAE-dextran-mediatedtransfection, electroporation (Chu et al., 1987, Nucleic Acid Res. 15,1311-1326), lipofection/liposome fusion (Feigner et al., 1987, Proc.Natl. Acad. Sci. USA 84, 7413-7417), particle bombardment (Yang et al.,1990, Proc. Natl. Acad. Sci. USA 87, 9568-9572), gene guns,transduction, infection (e.g. with an infective viral particle), andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001).

When the vector of the disclosure is defective, the infectious particlesare usually produced in a complementation cell line or via the use of ahelper virus, which supplies in trans the non-functional viral genes.For example, suitable cell lines for complementing adenoviral vectorsinclude the 293 cells (Graham et al., 1997, J. Gen. Virol. 36, 59-72) aswell as the PER-C6 cells (Fallaux et al., 1998, Human Gene Ther. 9,1909-1917) commonly used to complement the E1 function. Other cell lineshave been engineered to complement doubly defective adenoviral vectors(Yeh et al., 1996, J. Virol. 70, 559-565; Krougliak and Graham, 1995,Human Gene Ther. 6, 1575-1586; Wang et al., 1995, Gene Ther. 2, 775-783;Lusky et al., 1998, J. Virol. 72, 2022-2033; WO94/28152 and WO97/04119).The infectious viral particles may be recovered from the culturesupernatant but also from the cells after lysis and optionally arefurther purified according to standard techniques (chromatography,ultracentrifugation in a cesium chloride gradient as described forexample in WO 96/27677, WO 98/00524, WO 98/22588, WO 98/26048, WO00/40702, EP 1016700 and WO 00/50573).

The disclosure also relates to host cells which comprise the nucleicacid molecules, vectors or infectious viral particles of the disclosuredescribed herein. For the purpose of the disclosure, the term “hostcell” should be understood broadly without any limitation concerningparticular organization in tissue, organ, or isolated cells. Such cellsmay be of a unique type of cells or a group of different types of cellsand encompass cultured cell lines, primary cells and proliferativecells.

Host cells therefore include prokaryotic cells, lower eukaryotic cellssuch as yeast, and other eukaryotic cells such as insect cells, plantand higher eukaryotic cells, such as vertebrate cells and, with aspecial preference, mammalian (e.g. human or non-human) cells. Suitablemammalian cells include but are not limited to hematopoietic cells(totipotent, stem cells, leukocytes, lymphocytes, monocytes,macrophages, APC, dendritic cells, non-human cells and the like),pulmonary cells, tracheal cells, hepatic cells, epithelial cells,endothelial cells, muscle cells (e.g. skeletal muscle, cardiac muscle orsmooth muscle) or fibroblasts. Typical host cells include Escherichiacoli, Bacillus, Listeria, Saccharomyces, BHK (baby hamster kidney)cells, MDCK cells (Madin-Darby canine kidney cell line), CRFK cells(Crandell feline kidney cell line), CV-1 cells (African monkey kidneycell line), COS (e.g., COS-7) cells, chinese hamster ovary (CHO) cells,mouse NIH/3T3 cells, HeLa cells and Vero cells. Host cells alsoencompass complementing cells capable of complementing at least onedefective function of a replication-defective vector of the disclosure(e.g. adenoviral vector) such as those cited above.

The host cell of the disclosure can contain more than one nucleic acidmolecule, vector or infectious viral particle of the disclosure. Furtherit can additionally comprise a vector encoding a transgene, e.g. atransgene as described above. When more than one nucleic acid molecule,vector or infectious viral particle is introduced into a cell, thenucleic acid molecules, vectors or infectious viral particles can beintroduced independently or co-introduced.

Moreover, according to a specific embodiment, the host cell of thedisclosure can be further encapsulated. Cell encapsulation technologyhas been previously described (Tresco et al., 1992, ASAJO J. 38, 17-23;Aebischer et al., 1996, Human Gene Ther. 7, 851-860). According to saidspecific embodiment, transfected or infected eukaryotic host cells areencapsulated with compounds which form a microporous membrane and saidencapsulated cells can further be implanted in vivo. Capsules containingthe cells of interest may be prepared employing hollow microporousmembranes (e.g. Akzo Nobel Faser AG, Wuppertal, Germany; Deglon et al.1996, Human Gene Ther. 7, 2135-2146) having a molecular weight cutoffappropriate to permit the free passage of proteins and nutrients betweenthe capsule interior and exterior, while preventing the contact oftransplanted cells with host cells.

Still a further aspect of the present disclosure is a method forrecombinantly producing the fusion protein, employing the vectors,infectious viral particles and/or host cells of the disclosure. Themethod for producing the fusion protein comprises introducing a vectoror an infectious viral particle of the disclosure into a suitable hostcell to produce a transfected or infected host cell, culturing in-vitrosaid transfected or infected host cell under conditions suitable forgrowth of the host cell, and thereafter recovering said fusion proteinfrom said culture, and optionally, purifying said recovered fusionprotein. It is expected that those skilled in the art are knowledgeablein the numerous expression systems available for expression of thefusion proteins of the disclosure in appropriate host cells.

The host cell of the disclosure is typically produced bytransfecting/infecting a host cell with one or more recombinantmolecules, (e.g. a vector of the disclosure) comprising one or morenucleic acid molecules of the present disclosure. Recombinant DNAtechnologies can be used to improve expression of the nucleic acidmolecule in the host cell by manipulating, for example, the number ofcopies of the nucleic acid molecule within a host cell, the efficiencywith which the nucleic acid molecule is transcribed, the efficiency withwhich the resultant transcripts are translated, the efficiency ofpost-translational modifications and the use of appropriate selection.Recombinant techniques useful for increasing the expression of nucleicacid molecules of the present disclosure include, but are not limitedto, the use of high-copy number vectors, addition of vector stabilitysequences, substitution or modification of one or more transcriptionalregulatory sequences (e.g., promoters, operators, enhancers),substitution or modification of translational regulatory sequences(e.g., ribosome binding sites, Shine-Dalgamo sequences), modification ofnucleic acid molecule of the present disclosure to correspond to thecodon usage of the host cell, and deletion of sequences that destabilizetranscripts.

Host cells of the present disclosure can be cultured in conventionalfermentation bioreactors, flasks, and petri plates. Culturing can becarried out at a temperature, pH and oxygen content appropriate for agiven host cell. No attempts to describe in detail the various methodsknown for the expression of proteins in prokaryote and eukaryote cellswill be made here. In one embodiment, the vector is a plasmid carryingthe fusion-encoding nucleic acid molecule in operative association withappropriate regulatory elements. Typical host cells in use in the methodof the disclosure are mammalian cell lines, yeast cells and bacterialcells.

Where the fusion protein is not secreted outside the producing cell orwhere it is not secreted completely, it can be recovered from the cellby standard disruption procedures, including freeze thaw, sonication,mechanical disruption, use of lysing agents and the like. If secreted,it can be recovered directly from the culture medium. The fusion proteincan then be recovered and purified by well-known purification methodsincluding ammonium sulfate precipitation, acid extraction, gelelectrophoresis, reverse phase chromatography, size exclusionchromatography, ion exchange chromatography, affinity chromatography,phosphocellulose chromatography, hydrophobic-interaction chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography. The conditions and technology used topurify a particular fusion protein of the disclosure will depend on thesynthesis method and on factors such as net charge, molecular weight,hydrophobicity, hydrophilicity and will be apparent to those havingskill in the art. It is also understood that depending upon the hostcell used for the recombinant production of the fusion proteinsdescribed herein, the fusion proteins can have various glycosylationpatterns, or may be non-glycosylated (e.g. when produced in bacteria).In addition, the fusion protein may include an initial methionine insome cases as a result of a host-mediated process.

The fusion protein of the disclosure can be “purified” to the extentthat it is substantially free of cellular material. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the fusionprotein, even if in the presence of considerable amounts of othercomponents. In some uses, “substantially free of cellular material”includes preparations of the fusion protein having less than about 30%(by dry weight) other proteins (i.e., contaminating proteins), typicallyless than about 20% other proteins, more typically less than about 10%other proteins, or even more typically less than about 5% otherproteins. When the fusion protein is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

Terms

As used herein, the term “conjugate” refers to molecular entities joinedby covalent bonds or other arrangement that provides substantiallyirreversible binding under physiological conditions. For example, twoproteins may be conjugated together by a linker polymer, e.g.,polypeptide sequence, ethylene glycol polymer. Two proteins may beconjugated together by linking one protein to a ligand and linking thesecond protein to a receptor, e.g., streptavidin and biotin or anantibody and an epitope.

As used herein, the term “combination with” when used to describeadministration with an additional treatment means that the agent may beadministered prior to, together with, or after the additional treatment,or a combination thereof.

As used herein, “subject” refers to any animal, typically a humanpatient, livestock, or domestic pet.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity is reduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

As used herein, “amino acid sequence” refers to an amino acid sequenceof a protein molecule. An “amino acid sequence” can be deduced from thenucleic acid sequence encoding the protein. However, terms such as“polypeptide” or “protein” are not meant to limit the amino acidsequence to the deduced amino acid sequence, but include non-naturallyoccurring amino acids, post-translational modifications of the deducedamino acid sequences, such as amino acid deletions, additions, andmodifications such as glycolsylations and addition of lipid moieties.

The term “a nucleic acid sequence encoding” a specified polypeptiderefers to a nucleic acid sequence comprising the coding region of a geneor in other words the nucleic acid sequence which encodes a geneproduct. The coding region may be present in a cDNA, genomic DNA or RNAform. When present in a DNA form, the oligonucleotide, polynucleotide,or nucleic acid may be single-stranded (i.e., the sense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present disclosure may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

The term “recombinant” when made in reference to a nucleic acid moleculerefers to a nucleic acid molecule which is comprised of segments ofnucleic acid joined together by means of molecular biologicaltechniques. The term “recombinant” when made in reference to a proteinor a polypeptide refers to a protein molecule which is expressed using arecombinant nucleic acid molecule.

A “virus-like particle” refers to a particle comprising virion proteinsbut is substantially free of viral genetic material, e.g., viral RNA.Virus-like particles may contain viral proteins from different viruses.See e.g., Guo et al., Enhancement of mucosal immune responses bychimeric influenza HA/SHIV virus-like particles, Virology, 2003,313(2):502-13. Virus-like particles may contain lipid membranes and maybe constructed to express a variety of antigens on their particlesurface ether by expression in viral vectors use to create the particlesor by mixing the virus-like particle with an antigen or otherpolypeptide conjugated to a glycosylphosphatidyl-inositol anchor. Seee.g. Skountzou et al., J. Virol. 81(3):1083-94; Derdak et al., PNAS,2006, 103(35) 13144-13149; Poloso et al., Molecular Immunology, 2001,38:803-816.

As used herein, the article “a” or “an” is intended to refer to one ormore unless the context suggests otherwise.

EXPERIMENTAL Animals and Reagents

Wild-type female C57/BL6 (B6) mice, C57BL/6-Tg(TcraTcrb)1100Mjb/J(OT-I), C57BL/6-Tg(TcraTcrb)425Cbn/J (OT-II) used experimentally werebetween the age of 6-8 weeks, purchased from The Jackson Laboratory (BarHarbor Me.). Recombinant rIL-7, rGM-CSF, and their antibodies werepurchased from R&D systems (Minneapolis Minn.). Dulbecco's ModifiedEagle's Medium (DMEM), RPMI-1640, fetal bovine serum andPenicillin/Streptomycin were purchased from Wisent Technologies(Rocklin, Calif.). Cell separations were performed using EasySep kitsaccording to the manufacturer's instruction (StemCell Technologies,Vancouver BC Canada).

Fusokine Generation and Protein Modeling

The fusokine is composed of 2 entities. GM-CSF was amplified by PCR inorder to generate and subsequently cloned in frame at the N-terminuswith the cDNA encoding IL-7 into the expression vector pORF. As such,the chimeric transgene was expressed as a single open reading frame.HEK293 cells were seeded at 65-80% confluency and transientlytransfected using Polyfect (Qiagen, Mississauga, ON, Canada) andsupernatant was tested by western-blot. Three days later, thesupernatant was collected, concentrated using AMICONS Millipore(Cambridge, Ontario, Canada) and tested by western-blot. GIFT7expression levels were subsequently assessed using anti-IL-7 andanti-GM-CSF ELISA kit.

Human GIFT7 was constructed from the following nucleic acid sequence(SEQ ID NO: 3) ATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGC AGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCCAGTCCAGGAGTCACCGGTCAACATGTTCCATGTTTCTTTTAGGTATATCTTTGGACTTCCTCCCCTGATCCTTGTTCTGTTGCCAGTAGCATCATCTGATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACACTGA.

Murine GIFT7 was constructed from the following nucleic acid sequence(SEQ ID NO: 4) ATGTGGCT GCAGAATTTACTTTTCCTGGGCATTGTGGTCTACAGCCTCTCAGCACC CACCCGCTCACCCATCACTGTCACCCGGCCTTGGAAGCATGTAGAGGCCATCAAAGAAGCCCTGAACCTCCTGGATGACATGCCTGTCACGTTGAATGAAGAGGTAGAAGTCGTCTCTAACGAGTTCTCCTTCAAGAAGCTAACATGTGTGCAGACCCGCCTGAAGATATTCGAGCAGGGTCTACGGGGCAATTTCACCAAACTCAAGGGCGCCTTGAACATGACAGCCAGCTACTACCAGACATACTGCCCCCCAACTCCGGAAACGGACTGTGAAACACAAGTTACCACCTATGCGGATTTCATAGACAGCCTTAAAACCTTTCTGACTTCACCGGTAGGAGGGGCCAACATGTTCCATGTTTCTTTTAGATATATCTTTGGAATTCCTCCACTGATCCTTGTTCTGCTGCCTGTCACATCATCTGAGTGCCACATTAAAGACAAAGAAGGTAAAGCATATGAGAGTGTACTGATGATCAGCATCGATGAATTGGACAAAATGACAGGAACTGATAGTAATTGCCCGAATAATGAACCAAACTTTTTTAGAAAACATGTATGTGATGATACAAAGGAAGCTGCTTTTCTAAATCGTGCTGCTCGCAAGTTGAAGCAATTTCTTAAAATGAATATCAGTGAAGAATTCAATGTCCACTTACTAACAGTATCACAAGGCACACAAACACTGGTGAACTGCACAAGTAAGGAAGAAAAAAACGTAAAGGAACAGAAAAAGAATGATGCATGTTTCCTAAAGAGACTACTGAGAGAAATAAAAACTTGTTGGAATAAAATTTTGAAGGGCAGTATAT AA.

MSC-GIFT7 Implantation and C1498FFDsR Challenge

Whole bone marrow from femurs and tibias of CCL2^(−/−) C57BL/6 mice washarvested and placed in culture in complete medium until the appearanceof a homogeneous MSC polyclonal population. The mGIFT7 cDNA was clonedin the AP2 retroviral plasmid and retropracticles generated.Concentrated retroparticles were then used to gene modify CCL2^(−/−)C57BL/6 MSCs. Secretion and expression levels of GIFT7 by gene enhancedMSCs were analyzed by GMCSF and IL7 ELISA.

Cell Culture

Human embryonic kidney (HEK) 293 cell line was cultured in DMEM (WisentTechnologies) supplemented with 10% FBS (Wisent Technologies) and 100U/ml of Penicillin/Streptomycin (Wisent Technologies).RAW 264.7 cellline was purchased from ATCC and cultured in DMEM with 10% FBS and 100U/mL of penicillin/streptomycin. Primary mouse splenocytes and humanperipheral blood mononuclear cells (hPBMC) were cultured in RPMIsupplemented with 2 mM L-glutamine, 1 mM HEPES, 1 mM sodium pyruvate,0.05 mMβ-mercaptoethanol with 10% FBS. Spleen-derived T cells werecultured in complete splenocyte medium.

Apoptosis Analysis

For apoptosis assays, 106 CD8 T cells cultured for 48-72 hrs withequimolar concentrations of rGM-CSF, rIL-7, rGM-CSF/rIL-7, or GIFT7 wereanalyzed by PI and annexin-V staining to access percent apoptotic cells.

Western Blotting

For the detection of GIFT7, concentrated conditioned media from mock(PolyFect alone) or GMME3-transfected HEK 293 cells were denatured at90° C. for 5 minutes and separated on 4-20% SDS PAGE gel (Thermoscientific, Pittsburgh, Pa.). Immuno-blotting was performed withanti-IL-7 antibody and anti-GM-CSF antibody according to manufacturer'sguideline. For the study of the GMME3 biochemical response,1-2×106RAW264.7 cells or mouse spleen-derived primary T cells werestimulated with GIFT7, or cytokine controls in RPMI media for 20minutes. Cell lysates were prepared by using cell lytic M supplementedwith protease inhibitor and phosphatase inhibitor according tomanufacturer's instruction. Anti-phosphorylated or total STATS antibody(Cell Signaling Technology, Danvers, Mass.) was used in immuno-blotting.

Flow Cytometry and Intracellular Staining

Cells to be analyzed by fluorescence-activated cell sorter (FACS) wereharvested and resuspended in 10×10⁶ cells/ml. Samples were blocked withanti-FcRmAb 2.4G2 for 30 minutes and subsequently stained withfluorochrome-conjugated monoclonal antibody in Ca²⁺Mg²⁺ freephosphate-buffered saline (PBS) with 2% fetal bovine serum for 30minutes. Cells were washed twice with staining buffer and resuspended in1% PFA prior to analysis. Intracellular staining was performed withCytofix/Cytoperm Kit (BD) according to manufacturer's instruction. FACSantibodies were purchased from BD Pharmingen.

In Vitro Antigen Presentation Assay

Peritoneal macrophages were collected from lavage of the C57B/6 RBperitoneal cavity with 12 ml RPMI. Cells were cultured in 24-well platesand washed after 16 hours. Plastic adherent-macrophages were cultured inRPMI supplemented with 1 mg/ml of MOG 35-55 peptide (Sheldon BiotechCenter, McGill University) or chicken ovalbumin (Sigma) overnight. CD4⁺T cells were purified from the spleens of EAE mice with active diseaseor OT-II transgenic mice. CD8⁺ T cells were purified from the spleen ofOT-I transgenic mice. 5×105 purified T cells were cultured withantigen-pulsed, 1% paraformaldehyde-fixed macrophages in completesplenocyte medium. Alternatively, peritoneal-macrophages werepre-treated with 105 GMME3-activated B cells (BGMME3) for 48 hours.OTII-CD4⁺ or OT-I-CD8⁺ T cells were subsequently added to pre-treatedantigen-pulsed macrophages. Culture supernatant was collected after 72hours and inflammatory cytokine IFN-γ or IL17 were measured by ELISAwhen appropriate.

Human Ortholog of GIFT7 Leads to Enhanced T Cell Proliferation

Concentrated media conditioned by 2931 cells transfected withpORF-hGIFT7 was used to measure the expression and activity of hGIFT7.Immuno-blotting of conditioned media derived from hGIFT7-tranfectantprobed with α-hGMCSF or α-hIL7 antibody detected a bi-cistronic fusionprotein at roughly 60 kDa (FIG. 1). To test the bioactivity of GIFT7,pre-activated human PBMC were cultured with recombinant hIL7 or GIFT7.hGIFT7 induced a superior proliferative response compared to IL7 at aconcentration as low as 0.1 ug/mL in both CD4⁺ and CD8⁺ T cells. Thissuperior mitogenic effect was enhanced with increasing concentration(FIG. 6). The secreted hGIFT7 properly folded and leads to significantproliferative in T cells derived from PBMC.

Simian PBMC (sPBMC) Treated with hGIFT7 are Ki67^(hi) and PD1^(low)

The effect of hGIFT7 on simian T cells was investigated. Pre-activatedsPBMC were treated ex vivo with recombinant IL7 or GIFT7 (10 ng mL⁻¹ and50 ng mL⁻¹) for 3 or 7 days (FIG. 7A). GIFT7-treated sPBMC (both 10ngmL⁻¹ and 50 ngmL⁻¹) showed marked increase in absolute cell number assoon as day 3. On day 7, total cell number has been maintained (FIG.7B). In both CD4⁺ and CD8⁺ T cells, GIFT7 treatment increases total cellnumber after 3 or 7 days (FIG. 7C). Importantly, on day 7, the frequencyof Ki67⁺ T cells are significantly higher in both CD4⁺ (˜2 foldincrease) and CD8⁺ subset (˜50% increase), suggesting GIFT7 hasmaintained T cells in a proliferative state (FIG. 7D). Interestingly,GIFT7-mediated proliferative T cells have lower overall expression ofPD-1, especially in the CD8⁺ subset (FIG. 7E). Overall the data suggestthat the human ortholog of GIFT7 maintains simian T cell expansion andprevents exhaustion during the contraction phase (i.e. post-activation).

GIFT7 Leads to the Generation of Central Memory-Like T CellsPost-Activation

The effect of GIFT7 on T cells post-activation was examine to determinewhether GIFT7 leads to the expansion of memory-type T cells. CD3⁺purified T cells from WT C57B/L6 spleen were heterogeneous based on CD44and CD62L expression. αCD3/CD28 antibody-coated beads were used to mimicTCR stimulation. After 3 days, αCD3/CD28 beads were washed off and Tcell culture were supplemented with GIFT7 or monomeric cytokinecontrols. TCR stimulation leads to significant cell activation andproliferation (increase in FSC and SSC) with concurrent up-regulation ofCD44 and down-regulation of CD62L. Interestingly, GIFT7 signaling duringpost-activation maintained T cell population—preferentially enhanced theexpansion of CD8⁺ T cells—and induced the re-expression of CD62L,suggesting T_(GIFT7) acquire homing capacity to secondary lymphoidsystem (FIG. 8A). To analyze the phenotype between T_(GIFT7) andconventionally activated T cells (αCD3/CD28 in the presence of IL2), theexpression of a number of T cell differentiation markers were examined.CD8⁺ T_(GIFT7) were found to beCD62L^(hi)CD44^(hi)KLRG-1^(low)CD27^(hi); in particular, there was amarked upregulation of CD62L and CD27 on T_(GIFT7) compared toconventionally activated T cells (FIG. 8B). One of the hallmarks ofmemory-type T cells is their ability to persist in vivo. To determinewhether ex vivo generated T_(GIFT7) persist in vivo after reinfusion andexpand upon recall challenge, CD8⁺ T cells were purified fromOVA-specific OTI mice (CD45.2) and stimulated with αCD3/CD28 in thepresence of GIFT7 or IL2. OTI T_(GIFT7) or T_(IL2) were re-infused backto B6.SJL (CD45.1) mice. 3 weeks later ovalbumin was administered. Aftera week, splenocytes were analyzed for the co-expression of CD45.2 andCD8 to elucidate the persistence of the adoptive transferred T cells.There was a 10-fold increase in the survival of T_(GIFT7) compared toT_(IL2) after a recall challenge (FIG. 8C). GIFT7 signaling on T cellsduring the contraction phase not only rescues T cell pool but alsoexpand a population of CD8⁺ subset that exhibit a phenotype with betterin vivo fitness.

GIFT7-Mediated Anti-Tumor Effect In Vivo

CD8⁺ T cells were purified from OT1-trangenic mice and expanded in thepresence of GIFT7. The ex vivo expanded T_(GIFT7) cells weresubsequently re-infused back to OVA-expressing EG7-bearing mice. Twodoses of 8×10⁶ T_(GIFT7) adoptive transfer significantly delayed tumorprogression (FIG. 9A). Acute myelogenous leukemia C1498 cells cause CD8⁺immune effector failure via PD-1 up-regulation. Disease mortality isrelated to hepatocellular metastasis and subsequent immune dysfunction.See Zhang et al., Blood., 2009, 114(8):1545-1552. In order to test ifthe in vivo administration of GIFT7 can overcome AML-induced T cellexhaustion thereby decreasing tumor burden and improving overallsurvival, gene-enhanced autologous mesenchymal stromal cells (MSC) wereimplanted subcutaneously to act as protein delivery vehicle. It is aneo-organoid system that we have demonstrated to have led to sustainedprotein production detectable systemically. See Eliopoulos et al.,Cancer Res., 2008, 68(12):4810-4818. As such, mice challenged with 10⁶C1498 were co-transplanted with contigen-embedded MSC-GIFT7 or MSC-null(1.5×10⁶ cells/contigen). Overall, GIFT7 administration significantlyimproved survival with 50% of mice surviving a lethal dose of C1498challenge (FIG. 9B).

GIFT7 Acts as an IL7 Hyperagonist

To predict the possible binding mode of GIFT7 with IL7Rα, we in silicoaligned the IL7 portion of GIFT7 with the crystal structure of IL7 incomplex with IL7Rα. The GMCSF portion of GIFT7 is not in steric clashwith IL7Rα. N-terminal GMCSF may contribute to the binding of GIFT7 withIL7Rα. Immunoblotting demonstrates that GIFT7 is translated, secreted,and recognized by both α-GMCSF and α-IL7 antibodies (FIG. 1B). GIFT7stimulation leads to hyperphosphorylation of STATS downstream of IL7Rα(primary T cells) but not GMCSFR (RAW 264.7) compared to the combinationof its parental monomeric cytokines (FIG. 2). To determine the impact ofGIFT7-mediated hyperSTAT5 signalling, the surviving and proliferatingfraction of GIFT7-treated splenic T cells prestimulated in vitro withα-CD3/CD28-coated beads for 3 days were measured. TCR stimulationwithdrawal induced apoptosis in all control cytokine-treated T cellswhereas GIFT7 treatment led to significantly increased survivingfraction (FIG. 2). GIFT7-treated T cells resist contraction-inducedapoptosis, but also undergo substantial mitotic expansion.

GIFT7 Leads to DN Expansion In Vitro

Thymocytes were isolated from 6-8 week old mice and cultured in thepresence or absence of GIFT7 (10 ngmL⁻¹). Consistent with reportedliterature, dissociated thymus consist of approximately 4.5% DN, 4.2%SPCD8, 8% SPCD4, and 83% DP. Compared to control thymocyte cultures,GIFT7-treated thymocytes show significant DN and SPCD8 expansion in thelive cell fraction as early as 72 hours after stimulation. At later timepoint (day 7), the DN and SPCD8 fraction are 32% and 35% respectively inthe GIFT7-treated thymocyte culture. 7AAD staining also demonstratedGIFT7 protect DN and SPCD8 but not SPCD4 from apoptosis. This phenomenoncannot be recapitulated by GMCSF, IL7 or the combination of bothcytokines at equimolar concentrations. CFSE-pulsed DN cells showsignificant dilution after GIFT7 treatment in both TCRγδ⁻ and TCRγδ⁺compartments, demonstrating the mitogenic effect of GIFT7 on DN subsetin both αβ- and γδ-lineage (FIG. 4, panel C). GIFT7-expanded DNthymocytes increased expression of CD44 and granularity (FSC) but notCD25 or CD24, which corresponds to early thymic DN1 progenitors (FIG. 4,panel B).

To test the hypothesis that GIFT7 modulate early T cell differentiation,DN, SPCD4, SPCD8, and DP thymocytes were pre-sorted and subsequentlystimulated them with GIFT7 for 9 days. GIFT7 did not alter theexpression of CD4 and CD8 on pre-sorted cells. In all, these dataindicate that GIFT7—by provision of its hyperphosphoSTAT5 effect—leadsto the survival and expansion of CD4⁻CD8⁻CD25⁻CD24⁻CD44⁺ early thymicprogenitors and SPCD8 thymocytes in vitro without influencing theirdifferentiation programming.

GIFT7 Leads to Transient Thymic Hyperplasia in Immune-Competent YoungMice

The systemic effect of GIFT7 administration on thymic tissue in normal6-8 week old adult mice was investigated. Mice treated with GIFT7 or IL7were analyzed for thymic cellularity on day 7, 14 or 35 post-treatment.On day 7, both IL7 and GIFT7-treated mice showed significant increase intotal thymic cellularity, which continues to rise in GIFT7-treated groupbut starts to decline in the IL7 group by day 14 (237±51.3 v.s.123±25.6×106, p<0.05) (FIG. 10A). In particular, compared to IL7, GIFT7increased the percentage as well as the total number of DN1 cells by day7 (5.48±1.01 v.s. 2.74±1.07×106, p<0.05) (FIG. 10B). The ratio of thymicsubsets normalized between these two groups by day 14 despite anincrease in total thymocytes and the DP compartment (FIG. 10C). On day35 post-injection, total thymic cellularity in both GIFT7- andIL7-treated groups has declined to a level similar to that of prior totreatment (FIG. 10A). GIFT7 was able to overcome immune checkpoints,driving transient and reversible thymic hypertrophy. GIFT7 acts on theDN1 subset initially, which in turn contributed to the expansion of DPand total thymocytes at later stage even in an immune-replete state.This transient thymic-specific hypertrophic effect—splenic cellularityremains unchanged—demonstrates the gain of function of GIFT7 in vivo.

GIFT7 Corrects Age-Related Thymic Atrophy and Enhances Thymic Output

Whether GIFT7 can reverse age-associated thymic atrophy wasinvestigated. Aged mice were injected with 7 doses of IL7 or GIFT7 (5ug/Kg) every other day. On day 28 posttreatment, hematoxylin/eosin (H&E)staining on thymic tissues of IL7-treated aged mice shows extensiveadipocyte deposit (*), reduced cortical thymic tissue (cortex: medullaratio 2:1) with intact cortical medullary thymic lining (interruptedarrow) whereas GIFT7-treated mice exhibit minimal adipocyte deposits(*), hypercellularity in the cortex (cortex: medulla ratio 2.67:1) anddisrupted cortical-medullary lining with thymocytes infiltrating themedulla, indicating foci of proliferation (solid arrow) (FIG. 11A). Inaccordance with the histology data, GIFT7-treated mice havesignificantly higher number of total thymocytes (111±22, 68±14,60±4.6×106 p<0.05 for GIFT7-, IL7-treated, and untreated-groupsrespectively). The expansion is distributed in all four differentsubsets with DN exhibit the greatest fold difference (FIG. 11B). GIFT7administration enhances de novo T cell production in the periphery asmeasured by the mRNA level of single-joint TREC per TCRα in totalsplenic DNA (FIG. 11C). This indicates that GIFT7-mediated thymopoeisisundergo the normal process of T cell development, thus increasing thenumber of recent thymic emigrants in the periphery.

Regenerated CD44^(int) DN Function as Progenitors in GIFT7-Driven ThymicReconstitution

Further analysis of the DN compartment shows that exogenous GIFT7administration alters thymic composition in that CD44^(int)CD25⁻ DNsubset was significantly expanded in aged thyme (FIG. 12A). There is a4-fold increase in the number of CD44^(int) DN1 in addition to anexpansion in total DN1 and DN4 subsets (FIG. 12B). This indicates thatCD44^(int) DN1 is the most responsive subset to pharmacologicIL7-hypersignalling despite lower expression of IL7Rα (FIG. 12C). Thisdata indicates that CD44^(int)DN1 is the most responsive subset toIL7-mediated hypersignalling in vivo and is capable of partaking in Tcell neogenesis.

GIFT7 Treatment Enhances Anti-CMV CTL Response in Aged Mice

Latent cytomegalovirus (CMV) reactivation frequently correlates withimmune compromised state. Its clearance depends on functional T cellreconstitution in post bone marrow transplant (BMT) and sufficient naïveT cell output in the elderly. To analyze the effect of GIFT7 on acquiredanti-viral immunity in aged mice, IL7-, IL7⁺GMCSF-, GIFT7-, orPBSpreconditioned virus-naïve mice were challenged with a nonlethal doseof murine CMV (MCMV) 6 days post treatment. Mice were sacrificed 10 daysafter viral inoculation, and anti-MCMV T cells in the spleen wereenumerated (FIG. 13A). viral-specific cytotoxic T lymphocyte (CTL)immunity was measured by MCMV-peptide MHC tetramer⁺. Aged mice showrelative reduced MCMVspecific CTL response. GIFT7 treatmentsignificantly increased the frequency and number of MCMV-peptide MHCtetramer⁺ CD8⁺ T cells in the spleen (10.4±3.8 v.s. 2.9±1.4×10⁶ forGIFT7 and untreated respectively, p<0.05) (FIGS. 13B,C, and D). Thisindicates that GITF7-mediated thymic stimulation in aged mice lead toenhanced thymic output and adaptive cellular immunity against a definedviral pathogen.

What is claimed:
 1. A method of treating a viral infection comprisingadministering to a subject in need thereof an effective amount of apharmaceutical composition comprising a conjugate comprising a GM-CSFpolypeptide and an IL-7 polypeptide, and having the amino acid sequenceof SEQ ID NO: 1.